Anticonvulsant evaluation of clubbed indole-1,2,4-triazine derivatives: A synthetic approach

ACCEPTED MANUSCRIPT

Anticonvulsant evaluation of clubbed indole-1,2,4-triazine derivatives: A Synthetic

approach

Priya Ahuja, Nadeem Siddiqui*

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia Hamdard, Hamdard

Nagar, New Delhi-110062, India

ZONISAMIDE

O

N

LACOSAMIDE

O

VALPROIC ACID

OH

O

S

NH₂

O

H

N

O

N

(7a-f)

H

O

(8a-f)

(

6a-f)

O

S

VIGABATRIN

CARBAMAZEPINE

N

NH

2

O

HO

NH

2

O

OH

O

2

H N

O

2

H N

(

5a-f)

O

(9a-f )

R

N

NH

2

N

HN

S

(

4a-f)

NH₂

N

LAMOTRIGINE

NH₂

Cl

Indole C-3 substituted 5-amino-6-(5-substituted-2-phenyl-1H-indol-1-yl)-4,5-dihydro-1,2,4-

triazine-3(2H)-thione 5a-f, 6a-f, 7a-f, 8a-f and 9a-f were synthesized considering the

pharmacophoric elements and an array of functional groups necessary for anticonvulsant activity.

ACCEPTED MANUSCRIPT

Anticonvulsant evaluation of clubbed indole-1,2,4-triazine derivatives: A Synthetic

approach

Priya Ahuja, Nadeem Siddiqui*

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia Hamdard, Hamdard

Nagar, New Delhi-110062, India

Abstract

A series of thirty indole C-3 substituted 5-amino-6-(5-substituted-2-phenyl-1H-indol-1-yl)-4,5-

dihydro-1,2,4-triazine-3(2H)-thione 5a-f, 6a-f, 7a-f, 8a-f and 9a-f were synthesized to explore

prospective anticonvulsant agents. The derivative 1-(1-(5-amino-3-thioxo-2,3,4,5-tetrahydro-

1

,2,4-triazin-6-yl)-5-fluoro-2-phenyl-1H-indol-3-yl)ethanone (6b) had significant activity in

maximal electroshock test with minimal duration of limb extension (5.40 ± 0.61 sec) and

quantitative median dose of 7 mg/kg. In subcutaneous pentylenetetrazole screen 1-(5-amino-3-

thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)-5-fluoro-2-phenyl-1H-indole-3-sulfonamide

(7b)

increased the seizure latency to onset of clonus and was effective at a median dose of 35 mg/kg.

An in vitro radioligand binding assay on sodium channel and γ-amino butyric acid estimation

was also performed on active compounds to perceive the mechanistic procedure responsible for

it action.

Keywords: Triazine, Anticonvulsant, Quantification, Sodium channel, GABA

*

Corresponding author: Tel: +91 11 26059688 x 5639; Fax: +91 11 26059688 x 5307; Email

nadeems_03@rediffmail.com

1

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1

. Introduction

An idiopathic disorder of brain characterized by enduring predisposition resulting in sudden

onset of seizures and episodes of sensory, motor or autonomic phenomenon with or without loss

of consciousness is defined as epilepsy [1,2]. In the International League against Epilepsy and

International Bureau of Epilepsy it is depicted by the neurobiologic, cognitive, psychological,

and social consequences of the condition [3]. Despite the vast therapeutic arsenal of several

generations of antiepileptic drugs (AED’s), about 30% of adults are not free from seizures. The

condition gets worsened with infants having a series of encephalopathic syndromes [4-7]. Many

AED’s enlarge serious side effects that are increased when a lifelong medication is required [8].

Conversely the pharmacoresistant epilepsy [9], variable responses after epileptogenic brain

insults [10], comorbid medical conditions [11, 12], refractory epileptic population [13], limits the

attempts for treatment of antiepileptic patients. Moreover a major obstacle in the antiepileptic

drug development is the insufficient knowledge of the pathophysiology of epilepsy and modes of

action of existing drugs [14, 15]. It encourages the development of new antiepileptogenic [16]

drugs for its use earlier in the treatment chain. The several approaches towards this global

achievement in future has been the structural ‘improvements’ of already marketed drugs (anti-

ictal) or on discovering AED’s with novel mechanism of action (anti-epileptogenic) [17].

The indole ring has been found to be an integral nucleus in the past having diverse biological

activities such as anticancer, antiepileptic, anthelminthic, antidepressant, anti-inflammatory,

antihypertensive, carbonic anhydrase inhibitors etc [18-24]. Lamotrigine {3,5-diamino-6-(2,3-

dichlorophenyl)-1,2,4-triazine} is an antiepileptic drug associated with hypersensitivity reactions

which are thought to be an immunological response to metabolically generated drug-protein

adducts [25,26]. The o-dichlorophenyl moiety a metabolism-dependent hepatotoxicant, is a

potential site for bioactivation in rat and human liver that is oxidized to electrophilic arene oxide

that can be trapped by Glutathione. This epoxide is the primary cause for the hypersensitivity

reactions of lamotrigine. The diaminotriazine substituent is the dominant site of

biotransformation in most species (humans eliminate lamotrigine principally as N-glucuronides

in urine) [27, 28]. The 5-amino group is however the obligatory parameter, hence the 1,2,4-

triazine ring carrying the 5-amino group has been appended in our target compounds [29]. The

different substituents at the C-3 position whereas phenyl ring at C-2 of indole ring were

considered to have sufficient diverse electronic, steric and hydrophobic characters [30]. The C-3

vinyl, acetyl, sulphonamide, carboxylic and carboxamide substituents manifest the necessary

structural features of the prevalent AED’s [31-33]. They have also been ascertained as an

essential substituent in many previously synthesized anticonvulsants [34-36]. These elements

contribute as essential parameters to the pharmacophoric elements beside the lipophilic aryl ring,

hydrogen bonding domain of the triazine and distal aryl ring responsible for antiepileptic activity

(

Figure 1) [37,38]. The structural considerations of the indole ring and the 5-amino-1,2,4-triazine

ring of pharmacologically active AED lamotrigine gave impetus to synthesize their hybrid

molecules. These molecules were evaluated for anticonvulsant activity using maximal

2

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electroshock seizure (MES) test, subcutaneous pentylenetetrazole (scPTZ) test and minimal

motor impairment using rotarod test. In accord, the study of potency of these new derivatives on

two vital mechanisms such as γ-amino butyric acid (GABA) and sodium channel was also

accomplished. The mechanistic approach together with the data derived from the animal models

of seizures and epilepsy best describes the clinical efficacy profile of the drug.

2

. Result and Discussion

2

.1. Chemistry

The devised synthetic route for the preparation of the final compounds 5a-f, 6a-f, 7a-f, 8a-f and

a-f is delineated as follows. With the aim of developing cleaner and more benign process,

9

microwave assisted intramolecular electrophilic cyclization of substituted anilines and phenacyl

bromide was carried out via solvent-free Bischler indole synthesis. This greener synthesis of

compounds 1a-f prohibited the use of protecting groups, metallic reagents and catalysts used in

other synthetic methods of indoles. Further the drop-wise addition of chloroacetyl chloride in

presence of anhydrous potassium carbonate liberated the hydrochloric acid to generate the

compounds 2a-f. The use of thiosemicarbazide cyclized the N-chloroacetylated indoles to the

corresponding 3-thio-1,2,4-triazine derivatives 3a-f. These derivatives were then added in small

portions to a solution of potassium permanganate in dry liquid ammonia below 0 °C and purified

to render the lead compound of our series 4a-f (Scheme 1). The latter step was performed to

inoculate the 5-amino group onto the triazine ring. This 5-substituted-2-phenyl-N-1,2,4-triazine

indole derivatives 4a-f were further isosterized at the indole 3-position to generate the target

compounds. The presence of phenyl ring at the C-2 and the triazine ring at the N-1 position of

indole sterically hinder this position, leading to the attack by these isosteric substituents at the C-

3

position of π-excessive heteroarene indole ring [39]. The reaction with allyl bromide, acetic

anhydride, sulfuryl chloride in ammonia, trichloroacetic acid anhydride followed by sodium

hydroxide and further treatment of the corresponding carboxylic derivatives with thionyl

chloride in aqueous ammonia yielded the target compounds 5a-f, 6a-f, 7a-f, 8a-f and 9a-f under

their respective reaction conditions (Scheme 2). The compounds 5a-f was synthesized using

zinc-mediated barbier reaction while the compounds 6a-f utilized the Chichibabin synthetic

1

procedure for their synthesis. The structures of the compounds were confirmed by HNMR,

1

3

CNMR, IR and mass spectral data.

2

.2. Pharmacology

The initial evaluations for anticonvulsant activity were performed based on the Anticonvulsant

drug development program, Epilepsy Branch Neurological Disorders Program, NINDS [40]. The

maximal electroshock (MES) test was used as the experimental model to identify clinical

candidates that prevent the spread of tonic-clonic seizures and of partial convulsions with or

without secondary generalizations [41]. The subcutaneous pentylenetetrazole (scPTZ) test

3

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provided the clinically effective compounds against absence seizures besides elevating seizure

threshold [42]. The destitute from toxicity for the active compounds was established using the

minimal motor impairment-rotarod test [43]. The compounds were administered intraperitoneally

to mice at a dose of 30, 100 and 300 mg/kg at two pretreatment times (0.5 h and 4 h) prior to the

test. The reference drugs phenytoin (for MES and rotarod test) and ethosuximide (for scPTZ test)

were used as positive controls [44]. The negative control control group received 0.9 % saline (10

mL/kg).

In the preliminary screening all the newly synthesized compounds exhibited some degree of anti-

MES activity. The protection offered by these compounds was indicative of their

pharmacological ability against seizure spread at a certain dose level. The results obtained after

investigating the anticonvulsant activity of the synthesized compounds 5a-f, 6a-f, 7a-f, 8a-f and

9

7

a-f are summarized in table 1. An instill into these outcomes showed that compounds 6b, 6d,

a, 7b, 8d and 9c were more effective at a dose of 30 mg/kg at both reported time intervals. It

depicts the quick onset and prolonged anticonvulsant potential of these derivatives at minimum

dose comparable to the reference drug phenytoin. It is explicit of the remarkable activity

displayed by them at this preliminary level. A similar kind of rapid onset at low dose and long

lasting effects (4 h) but at a higher dose of 100 mg/kg were shown by the compounds 5d, 6a, 7d,

8

c, 8f and 9b. In the allyl substituted indole-3-derivatives 5a-f, the electron withdrawing

substituents trifluoromethyl (CF ) and chloro were more effective than the electron donating

substituents thiomethyl (SCH ) and methoxy (OCH ). The analysis of the results for electron

3

withdrawing substituents is suggestive of the greater size of the CF and Cl than the

corresponding NO and F, responsible for their anticonvulsant activity. An almost reverse of the

2

activity of the compounds 5a-f was depicted by the carboxamide derivatives 9a-f with nitro

derivative (9c) being the most effective at the minimum dose of 30 mg/kg at both the time

intervals. This factual observation can be somewhat attributed to the negative inductive effect as

well as resonance stabilization shown by the nitro group that helps it to effectively bind to the

receptor site. Moreover compounds 6a-f containing acetyl substitution at the indole C-3 showed

that greater electronegative fluorine substituents (6b, 6d) were more effective than the

corresponding chloro derivatives. The cessation against electroshock protection was observed in

nitro derivative (6c) at delayed absorption interval (4 h). On the other hand, among the

sulphonamide substituted indole derivatives 7a-f, halogens (Cl and F) and its hydrocarbon

3

derivative (CF ) indicated good protection than the corresponding electron donating substituents

(

OCH and SCH ) derivatives against electrically induced seizures. In these derivatives also the

3

nitro derivative (7c) did not showed activity at 4 h. This can accord to the quick metabolism of

the nitro derivative in presence of the acetyl and sulphonamide groups. Furthermore in the indole

derivatives terminating with carboxylic acid 8a-f at position three, the absorption by halogens

(Cl and F) was at a higher dose following cessation of activity at longer duration. Amongst all

the methoxy derivatives, the compounds 6e, 7e, 8e and 9e demonstrated anticonvulsant potential

at a dose of 100 mg/kg at both the reported intervals irrespective of the substituent at the third

4

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position of the indole ring. The methoxy substitution on the indole-3-allyl derivative (5e),

however, was active at higher dose of 300 mg/kg followed by cessation of activity at 4 h

duration. The CF derivatives at indole 5-position showed high efficacy in conjunct with the

3

strong electron withdrawing substituents (COCH and COOH) followed by lesser electron

3

withdrawing substituents (allyl and SO NH ) at the indole 3-position. Replacement of electron

2

withdrawing substituents by electron donating substituents (CONH ) at the indole 3-position,

2

leads to decline in the activity of CF substituted derivatives.

3

The general anti-MES protective capability of the synthesized compounds can be demonstrated

as:

5

6

7

8

9

a-f: CF > Cl> SCH > NO > F ≡ OCH

3 3 2

3

2

a-f: F ≡ CF > Cl> OCH > NO > SCH

3

2

a-f: Cl ≡ F> CF

> OCH

3

> SCH > NO

3

a-f: CF > NO ≡ SCH > OCH > Cl ≡ F

3

2

3

a-f: NO

2

> F> OCH

3

3 3

≡ SCH > Cl> CF

In the scPTZ screen, most of the compounds showed varying degree of protection and some

derivatives were devoid of any activity. The strongest anticonvulsant potential was exhibited by

the compounds 6b, 7b and 8c. Furthermore, the compounds 5c, 6c, 7c, 7f, 8b and 9b had an

equipotent effect to that of the reference drug ethosuximide. In the allyl substituted indole

derivative 5a-f, the tendency to protect chemically induced seizures was same as observed for

the carboxamide derivatives 9a-f in the MES test. On continuation of analyzing the outcomes of

the acetyl derivatives 6a-f, interestingly we found their activity trend similar to its MES

protection except the interchange of the nitro with the trifluoromethyl derivative, former being

3 3

more active than the latter. The CF and SCH substituted compounds showed equipotent activity

in both the acetyl (6d, 6f) and carboxylic acid (8d, 8f) derivative. A drift in the activity of the

thiomethyl substituted indole-3-sulphonamide derivative (7f) was seen compared to the acetyl

derivatives making it equally efficacious to the corresponding nitro derivative (7c). Furthermore

a likewise sequence of scPTZ protection was observed for indole-3-carboxylic acid derivative

except the nitro substituent. The nitro derivative (8c) was found to be more effective than the

electron withdrawing fluoro derivative (8b). This can be attributed to the additive resonance

stabilization effect exhibited by this group compared to only –I effect of fluorine. In the indole-3-

carboxamide terminal derivatives, the maximum efficacy was observed for the electron

withdrawing substituents followed by the releasing groups. However, the chloro derivative (9a)

lacked anticonvulsant activity against pentylenetetrazole induced seizures which can be

envisaged to its lower electron affinity compared to other electron withdrawing groups.

We noticed that all the methoxy substituted derivatives 5e, 6e, 7e and 8e depicted the similar

anticonvulsant potential at a dose of 300 mg/kg at both the intervals irrespective of the

substitution at the indole 3-position. It corresponds to the similar behavior shown by the methoxy

derivatives against electrically induced seizures in the MES test. The fluoro (8b and 9b) and the

thiomethyl derivatives (8f and 9f) demonstrated alike activities in conjunct with carboxylic acid

5

ACCEPTED MANUSCRIPT

and its amide at the third position of indole ring. Likewise the chloro derivative 6a, 7a and 8a

had same anti-scPTZ activity but at the maximum dose. On the other hand the nitro derivatives

gave protection against the PTZ induced seizures at the same dose in the allyl (5c), acetyl (6c)

and sulphonamide derivatives (7c) too. The chloro substitution was found to be ineffective

whence in conjuction with the allyl (5a) and carboxamide (9a) moieties at indole 3-position. In

all the synthesized compounds the greater electronegative fluoro derivatives were more effective

than the corresponding chloro compounds as well as the sterically hindered trifluoromethyl

substituted compounds.

The general anti-scPTZ protective capability of the synthesized compounds can be demonstrated

as:

5

6

7

8

9

a-f: NO > F ≡ OCH > SCH > Cl ≡ CF

2

3

a-f: F> NO

2

> Cl ≡ OCH

> CF ≡ SCH

3 3 3

a-f: F> NO ≡ SCH > Cl ≡ OCH > CF

2

3

a-f: NO

2

> F> Cl ≡ OCH

3

> CF

3

≡ SCH

3

a-f: F> NO ≡ CF > SCH > OCH > Cl

2

3

All the active compounds were found to be non-neurotoxic at the maximum dose at bith the

reported time intervals.

The potent compounds in the preliminary anticonvulsant screen were further assessed against

seizure challenges upon oral administration. The duration of limb extension formed the basis of

MES screen [45] whereas seizure latency to onset of clonus [46] was defined as the parameter

for the anti-scPTZ efficacy. The test compounds were administered at a dose of 100 mg/kg, 60

min before the injection of PTZ (85 mg/kg, sc) and MES test (150 mA, 0.2 s). The latency time

was observed for 14 min in scPTZ test. Phenytoin (30 mg/kg, oral) and sodium valproate (200

mg/kg, p.o) [45,47] were used as reference standard for MES and scPTZ anticonvulsant

evaluation. The test and the reference compounds were prepared in 0.5% sodium

carboxymethylcellulose. The results were analyzed using one way analysis of variance

(ANOVA) followed by Dunnett’s method and expressed as means ± SEM. The absence of hind

limb tonic extension during electroshock induced seizures and non-mortality after PTZ

administration was considered as protection. The record of the MES test suggested that

compound 6b was the most effective of the series. It significantly decreased the duration of limb

extension compared to control (p < 0.01) besides manifesting 100% protection in the

experimental group (Table 2). 7a and 7b caused a decrease in the duration of tonic extension but

the % protection reduced to 66% and 83.3% respectively. The remaining examined derivatives

offered moderate protection. In the scPTZ test the compounds 6b and 7b delayed the onset of

myoclonic jerks of forelimbs with 7b exceeding the latency period of the reference sodium

valproate. The anti-scPTZ protection shown by them was however less than that of the standard.

Conversely the compound 8c had a non-significant impact on the latency interval in this

experimental evaluation. All the efficacious compounds in this oral screen were non-neurotoxic

6

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at the dose of administration. Hence the compound 6b in MES test and 7b in scPTZ test were

crowned as the most effective of all the tested derivatives after oral administration.

The significantly active compounds in the primary MES and scPTZ test on i.p and oral

administration crusaded in the need for quantitative studies [48]. Table 3 reports the median

effective doses (ED ) for both the screens and median doses for neurological impairment (TD )

5

0

50

in mice after intraperitoneal administration. The pharmacological data of MES test revealed

compound 6b as the most potent with an ED of 7 mg/kg at the time of peak effect. However the

5

0

TD of 6b producing deficits in motor coordination in 50% of animals was found to be 290

5

0

mg/kg, much greater than the standard drug phenytoin (TD = 66 mg/kg) [49]. The compound

5

0

7

b though having equal ED₅₀to phenytoin, outplayed the median toxic dose to 225 mg/kg

thereby increasing its protective index. The compound 7a provided moderate efficacy. In the

scPTZ test, 7b was quantified as the most potent compound with nearly one-fourth the effective

dose of the standard drug ethosuximide. Moreover the median toxic dose of 7b (TD₅₀= 225

mg/kg) though less than standard (TD50 = 440.8 mg/kg) yet the overall protective index

(

TD /ED ) of 7b (PI = 6.43) exceeded that of the standard (PI = 3.39). 6b gave nearly equal

50 50

protection to the standard.

To infer the mechanistic approach of the synthesized derivatives, the active compounds were

neurochemically investigated for their effect on GABA levels in whole rat brain. It was

performed on a group of six rats each taking clobazam (30 mg/kg, p.o) as reference [50]. The test

compounds were administered intraperitoneally in the first study to a set of animals at a dose of

1

00 mg/kg. The brains were removed after 2 h of drug administration and the levels of GABA

were estimated in the whole brain. The values were expressed as means ± SEM. In the second set

of animals the study was conducted for seven days. The test drugs were given orally for this

period and the GABA levels were estimated on removal of brain after 4 h of seventh day. The

findings are mentioned in the table 4. All the test compounds significantly increased the GABA

levels in comparison to the control at both the reported time intervals. The values were compared

using student’s t test. It signifies their anticonvulsant potential as increase in GABA is inferred as

a cardinal factor in the anticonvulsant drug discovery.

An insight into the physiological and biochemical events in clinical condition shadows the

underlying molecular mechanisms in epilepsy. Many AED’s like phenytoin, carbazepine and

lamotrigine act via prolonging inactivation of voltage dependent sodium channels. Thus sodium

channel is a potential site for pharmacological action of anticonvulsants. For a drug hunter, the

valuable leads obtained via in vivo testing of compounds in MES test provided the basis for

deducing in vitro binding affinity for neuronal sodium channels of these compounds. The

3

displacement of [ H]BTX from neurotoxin site 2 of voltage-gated sodium channels in rat

forebrain tissue provides an indication of the selective pharmacological modulation by these

compounds [51]. The outcomes of the three compounds 6b, 7a and 7b are mentioned as IC₅₀

(ꢀM) and the % inhibition at a concentration of 100 ꢀM in table 5. Amongst the three screened

compounds, the compound 6b blocked the [3H]BTX-B binding on statistically significant

7

ACCEPTED MANUSCRIPT

fraction of sodium channel receptor site by 50.40% at 100 ꢀM. It was found to behave similarly

to the standard drug phenytoin with 49.61% inhibition, used as a positive control. The IC₅₀

values of 6b, 7a and 7b are 108 ± 5 ꢀM, 300 ± 2 ꢀM and 220 ± 6 ꢀM compared to 126 ± 7 ꢀM

for phenytoin (Figure 2). Hence the radioligand binding result of 6b boosts the confidence in

rational design of the targeted compound as neuronal sodium channel blocker.

3

. Conclusion

A series of thirty 5-amino-6-(3,5-disubstituted-2-phenyl-1H-indol-1-yl)-4,5-dihydro-1,2,4-

triazine-3(2H)-thione 5a-f, 6a-f, 7a-f, 8a-f and 9a-f were synthesized by us envisaged as

potential anticonvulsants. The rationale employed behind these derivatives was the addition of

the structural elements of the established drugs to the indole-N-1,2,4-triazine nucleus such as to

improve the anticonvulsant potential. To derive a mechanistic procedure responsible for its

action the study on sodium and GABA channel was also carried out based on its structural

resemblance to the reference AED’s. After the preliminary studies in both the MES and scPTZ

models on intraperitoneal and oral administration, the active compounds were challenged against

the quantitative evaluation and GABA estimation in rat whole brain. In vitro screening for

3

sodium channel activity by [ H]BTX radioligand assay was also performed for the compounds

having activity in MES test. The compound 6b was the most active of the series possessing a

minimal ED₅₀and a very high TD₅₀values, reducing acute mortality and duration of limb

extension, while increasing the GABA level as well as the % inhibition of sodium channel. The

compound 7b had significant activity in the scPTZ screen decreasing the frequency of seizures

besides increasing the latency to spontaneous recurrent seizures. The outcomes of this viable

schematic strategy experiments stimulated the molecular modeling and quantitative structure

activity relationship tools to be applied on these compounds. They are underway and will be

reported in due course.

4

. Experimental

4

.1. Chemistry

All reagents were used as purchased from commercial suppliers without further purification.

Thin layer Chromatography (TLC) was performed with Silica gel 60 F₂₅₄TLC aluminium sheet

(Merck, KGaA, Darmstadt, Germany) using Toluene: Ethyl acetate: Formic acid (5:4:1) as

eluents. Ashless Whatmann No. 1 filter paper was used for vacuum filtration. Melting points

were determined by using open capillary tubes in a Hicon melting point apparatus and are

uncorrected. The purity of the compounds was determined through elemental analysis. Elemental

data of C, H and N was found in accordance with ± 0.4% of the theoretical value respectively as

determined by Vario EL III CHNS Elementar. The Infrared Spectra of compound was recorded

on Bio-rad FTS-135 spectrophotometer using KBr pellets. The abbreviated forms mentioned in

1

13

the IR interpretation are as follows Ar, aryl; br, broad; str, stretching. HNMR and CNMR

8

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spectra in DMSO-d

6

/CDCl

3

solutions were respectively recorded at 400 and 100 MHz with

Bruker 400 Ultra shield TM NMR spectrometer (400 MHz) using TMS [(CH ) Si] as internal

3

4

standard. Splitting patterns are designated as follows: s, singlet; d, doublet; t, triplet; m,

multiplet; brs, broad singlet; dd, double doublet. Chemical shifts (δ) values are given in parts per

million (ppm). J values are given in Hz. The Mass Spectra were recorded on a Waters Micro-

mass ZQ 2000 Spectrophotometer.

4

.1.1. General procedure for the preparation of compound (1a-f)

Phenacyl bromide (1 mmol) was stirred with para-substituted aniline (2 mmol) at room

temperature without any base to neutralize the liberated hydrogen bromide. The mixture was

kept at room temperature with occasional stirring for 3 h. To the solid mixture, containing N-

phenacyl aniline and anilinium hydrobromide, was added 3-4 drops of dimethyl formamide and

the mixture was irradiated in a microwave oven at 600 W for 1 min for the cyclization. After

completion of the reaction, the mixture was treated as below to give the pure 2-arylindoles. After

completion of the reaction the mixture was loaded onto a silica gel column and pure 2-

arylindoles were obtained by chromatography, eluting with a gradient starting from 9:1

petroleum ether-ethyl acetate. Alternatively, the mixture could also be extracted with ethyl

acetate, washed with water, dried and evaporated before chromatography.

4

.1.2. General procedure for the preparation of compound (2a-f)

The indole derivatives 1a-f (0.05 mol) and anhydrous potassium carbonate (0.05 mol) were

dissolved in 100 mL absolute toluene. To this refluxing solution, there was slowly added drop-

wise chloroacetylchloride (6.4 mL, 0.08 mol) dissolved in toluene (20 mL), after which the

reaction mixture was refluxed for another hour. The residue was re-crystallized from ethanol

after distillation of the solvent and the excess of chloroacetylchloride.

4

.1.3. General procedure for the preparation of compound (3a-f)

A mixture of the chloroacetylated derivatives 2a-f (0.01 mol) and thiosemicarbazide (0.01 mol)

in 10% ethanolic sodium hydroxide solution (20 mL) was refluxed for 8 h. The reaction mixture

was cooled and poured onto ice/cold water acidified with few drops of hydrochloric acid. The

formed precipitate was filtered off, dried and re-cystallized from chloroform.

4

.1.4. General procedure for the preparation of compound (4a-f)

3

-thio-1,2,4-triazine derivatives 3a-f (10 mmol) was added in small portions to a solution of

potassium permanganate (2.0 g) in dry liquid ammonia (100 mL) below 0 °C and stirred. The

mixture was kept at this condition for 15 minutes. Ammonia was then evaporated and the residue

was extracted with hot isopropanol (50 mL). Removal of the solvent and re-crystallization from

ethanol gives the pure product.

4

.1.5. General procedure for the preparation of compound (5a-f)

9

ACCEPTED MANUSCRIPT

A mixture of indoles 4a-f (5 mmol), allyl bromide (10 mmol), zinc powder (10 mmol) in

Tetrahydrofuran (10 mL) was stirred at room temperature for 5-6 h. After completion of the

reaction as indicated by TLC, the reaction mixture was quenched with saturated ammonium

chloride (15 mL) and extracted with ethyl acetate (2×15 mL). Evaporation of the solvent

followed by purification on silica gel using ethyl acetate-hexane (9:5) as solvent afforded pure

allyl indole derivatives 5a-f.

4

.1.5.1.

6-(3-allyl-5-chloro-2-phenyl-1H-indol-1-yl)-5-amino-4,5-dihydro-1,2,4-triazine-

(2H)-thione (5a)

3

-1

Yield: 85%; m.p. 185-188 ºC; IR (KBr) υ (cm ): 3095 (C-H Ar str), 2940 (C-H str), 1615 (N-

C=N str), 1596, 1475 (C=C str), 1550 (Het. Triazine str), 1518 (C=C Ar str), 1040 (C-Cl str);

1

6 2 2

HNMR (300 MHz, DMSO-d ) δ: 2.12 (m, 1H, CH=CH , J = 1.6 Hz), 3.10 (d, 2H, CH , J = 6.7

Hz), 3.97 (s, 1H, 5”CH), 4.23 (brs, 2H, 5”NH , D O exchangeable), 5.05 (d, 1H, H =CH , J =

2

A

2

1

.6 Hz), 5.34 (dd, 1H, H

Hz, J = 8.5 Hz), 7.22-7.39 (m, 3H, 3’,4’,5’CH, J = 4.8 Hz, J = 8.5 Hz), 7.71 (d, 2H, 2’,6’CH, J =

.28 Hz), 7.76 (d, 1H, 7CH, J = 8.5 Hz), 8.21 (s, 1H, 4CH), 9.03 (brs, 1H, 4”NH, D

B 2

=CH , J = 0.8 Hz, J = 1.6 Hz, J = 6.7 Hz), 7.09 (dd, 1H, 6CH, J = 2.5

7

2

O

1

3

exchangeable), 12.21 (brs, 1H, 2”NH, D O exchangeable); CNMR δ (ppm) (DMSO-d , TMS):

2

6

3

1

5.2 (CH

2

), 66.4 (5”CH), 109.1 (C-3), 110.4 (C-7), 114.7 (CH=CH

2

), 119.3 (C-4), 121.0 (C-6),

24.2 (2’,6’CH), 126.1 (C-5), 127.2 (4’CH), 127.7 (CH-C (q) fused carbon), 128.5 (3’,5’CH),

30.8 (C-2), 133.4 (1’CH), 134.5 (C-N (q) fused carbon), 135.1 (CH=CH ), 160.9 (6”CH), 185.2

2

(

C=S); MS (70 ev): m/z = 396.35 (M+1); Anal. Calcd. for C H ClN S (395.91): C, 60.67; H,

20 18 5

4

.58; N, 17.69, Found, C, 60.74; H, 4.52; N, 17.60.

4

.1.5.2.

6-(3-allyl-5-fluoro-2-phenyl-1H-indol-1-yl)-5-amino-4,5-dihydro-1,2,4-triazine-

(2H)-thione (5b)

3

-1

Yield: 88%; m.p. 178-183 ºC; IR (KBr) υ (cm ): 3090 (C-H Ar str), 2945 (C-H str), 1623 (N-

C=N str), 1598, 1471 (C=C str), 1551 (Het. Triazine str), 1525 (C=C Ar str), 1154 (C-F str);

1

HNMR (300 MHz, DMSO-d ) δ: 2.14 (m, 1H, CH=CH , J = 1.3 Hz), 3.13 (d, 2H, CH , J = 6.5

6

2

Hz), 3.99 (s, 1H, 5”CH), 4.22 (brs, 2H, 5”NH , D O exchangeable), 5.08 (d, 1H, H =CH , J =

2

A

2

1

.6 Hz), 5.32 (dd, 1H, H =CH , J = 0.9 Hz, J = 1.6 Hz, J = 6.5 Hz), 7.12 (dd, 1H, 6CH, J = 2.1

B 2

Hz, J = 8.4 Hz), 7.23-7.37 (m, 3H, 3’,4’,5’CH, J = 4.8 Hz, J = 8.7 Hz), 7.68 (d, 2H, 2’,6’CH, J =

7

.28 Hz), 7.84 (d, 1H, 7CH, J = 8.0 Hz), 8.40 (s, 1H, 4CH), 9.05 (brs, 1H, 4”NH, D O

2

1

3

2 6

exchangeable), 12.25 (brs, 1H, 2”NH, D O exchangeable); CNMR δ (ppm) (DMSO-d , TMS):

3

1

5.5 (CH ), 66.1 (5”CH), 109.5 (C-3), 110.7 (C-7), 111.3 (C-4), 114.4 (CH=CH ), 117.2 (C-6),

2 2

24.6 (2’,6’CH), 127.0 (4’CH), 127.8 (CH-C (q) fused carbon), 128.1 (3’,5’CH), 130.3 (C-2),

33.2 (1’CH), 134.7 (C-N (q) fused carbon), 135.6 (CH=CH ), 160.6 (6”CH), 161.5 (C-5), 184.8

2

5

(C=S); MS (70 ev): m/z = 380.10 (M+1); Anal. Calcd. for C20H18FN S (379.45): C, 63.31; H,

4

.78; N, 18.46, Found, C, 63.39; H, 4.72; N, 18.39.

1

0

ACCEPTED MANUSCRIPT

4

.1.5.3.

6-(3-allyl-5-nitro-2-phenyl-1H-indol-1-yl)-5-amino-4,5-dihydro-1,2,4-triazine-

3

(2H)-thione (5c)

-1

Yield: 74%; m.p. 198-202 ºC; IR (KBr) υ (cm ): 3096 (C-H Ar str), 2948 (C-H str), 1630 (N-

C=N str), 1600, 1470 (C=C str), 1563 (Het. Triazine str), 1531 (C=C Ar str), 1325 (NO str);

2

1

HNMR (300 MHz, DMSO-d ) δ: 2.17 (m, 1H, CH=CH , J = 1.6 Hz), 3.11 (d, 2H, CH , J = 6.5

6

2

Hz), 3.97 (s, 1H, 5”CH), 4.19 (brs, 2H, 5”NH , D O exchangeable), 5.06 (d, 1H, H =CH , J =

2

A

2

1

.8 Hz), 5.35 (dd, 1H, H =CH , J = 0.8 Hz, J = 1.8 Hz, J = 6.6 Hz), 7.15 (dd, 1H, 6CH, J = 2.2

B 2

Hz, J = 8.2 Hz), 7.27-7.38 (m, 3H, 3’,4’,5’CH, J = 4.9 Hz, J = 8.9 Hz), 7.70 (d, 2H, 2’,6’CH, J =

.30 Hz), 7.95 (d, 1H, 7CH, J = 8.4 Hz), 8.71 (s, 1H, 4CH), 9.02 (brs, 1H, 4”NH, D O

7

2

1

3

exchangeable), 12.22 (brs, 1H, 2”NH, D O exchangeable); CNMR δ (ppm) (DMSO-d , TMS):

2

6

3

5.1 (CH ), 66.3 (5”CH), 108.5 (C-7), 109.2 (C-3), 114.7 (CH=CH ), 116.7 (C-6), 124.5

2 2

(

2’,6’CH), 127.1 (4’CH), 127.3 (C-4), 127.8 (CH-C (q) fused carbon), 128.4 (3’,5’CH), 130.6

C-2), 132.4 (C-5), 133.8 (1’CH), 134.9 (CH=CH ), 140.5 (C-N (q) fused carbon), 161.0

2

18 6 2

6”CH), 185.2 (C=S); MS (70 ev): m/z = 407.00 (M+1); Anal. Calcd. for C20H N O S (406.46):

C, 59.10; H, 4.46; N, 20.68, Found, C, 59.15; H, 4.52; N, 20.62.

4

.1.5.4.

6-(3-allyl-2-phenyl-5-(trifluoromethyl)-1H-indol-1-yl)-5-amino-4,5-dihydro-

1

,2,4-triazine-3(2H)-thione (5d)

-1

Yield: 79%; m.p. 210-215 ºC; IR (KBr) υ (cm ): 3099 (C-H Ar str), 2941 (C-H str), 1620 (N-

C=N str), 1602, 1475 (C=C str), 1546 (Het. Triazine str), 1535 (C=C Ar str), 1164 (C-F str);

1

HNMR (300 MHz, DMSO-d ) δ: 2.11 (m, 1H, CH=CH , J = 1.4 Hz), 3.08 (d, 2H, CH , J = 6.8

6

2

Hz), 3.95 (s, 1H, 5”CH), 4.25 (brs, 2H, 5”NH

2

, D

2

O exchangeable), 5.06 (d, 1H, H

A 2

=CH , J =

1

3

2

.7 Hz), 5.30 (dd, 1H, H =CH , J = 0.6 Hz, J = 1.7 Hz, J = 6.7 Hz), 7.22-7.35 (m, 3H,

B 2

’,4’,5’CH, J = 4.7 Hz, J = 8.6 Hz), 7.54 (dd, 1H, 6CH, J = 2.0 Hz, J = 8.0 Hz), 7.68 (d, 2H,

’,6’CH, J = 7.26 Hz), 7.87 (d, 1H, 7CH, J = 8.1 Hz), 8.86 (s, 1H, 4CH), 9.04 (brs, 1H, 4”NH,

1

3

D O exchangeable), 12.20 (brs, 1H, 2”NH, D O exchangeable); CNMR δ (ppm) (DMSO-d ,

2

6

TMS): 35.3 (CH ), 66.4 (5”CH), 109.4 (C-3), 111.5 (C-7), 114.8 (CH=CH ), 115.1 (C-6), 117.4

2

(

C-4), 124.0 (2’,6’CH), 124.4 (CF ), 125.5 (C-5), 127.7 (4’CH), 128.0 (CH-C (q) fused carbon),

3

1

28.4 (3’,5’CH), 130.5 (C-2), 133.1 (1’CH), 134.6 (C-N (q) fused carbon), 135.3 (CH=CH₂),

61.0 (6”CH), 185.5 (C=S); MS (70 ev): m/z = 430.09 (M+1); Anal. Calcd. for C H F N S

2

1

18

3

5

(429.46): C, 58.73; H, 4.22; N, 16.31, Found, C, 58.78; H, 4.17; N, 16.35.

4

.1.5.5.

6-(3-allyl-5-methoxy-2-phenyl-1H-indol-1-yl)-5-amino-4,5-dihydro-1,2,4-

triazine-3(2H)-thione (5e)

Yield: 76%; m.p. 203-208 ºC; IR (KBr) υ (cm ): 3092 (C-H Ar str), 2955 (C-H str), 1618 (N-

-1

C=N str), 1592, 1475 (C=C str), 1560 (Het. Triazine str), 1533 (C=C Ar str), 1252 (C-O-C str);

1

6 2 2

HNMR (300 MHz, DMSO-d ) δ: 2.10 (m, 1H, CH=CH , J = 1.5 Hz), 3.12 (d, 2H, CH , J = 6.6

Hz), 3.68 (s, 3H, OCH ), 3.99 (s, 1H, 5”CH), 4.21 (brs, 2H, 5”NH , D O exchangeable), 5.07 (d,

3

2

1

H, H

A

=CH

2

B 2

, J = 1.5 Hz), 5.34 (dd, 1H, H =CH , J = 0.7 Hz, J = 1.8 Hz, J = 6.5 Hz), 7.07 (dd,

H, 6CH, J = 2.2 Hz, J = 8.3 Hz), 7.26-7.37 (m, 3H, 3’,4’,5’CH, J = 4.8 Hz, J = 8.4 Hz), 7.69 (s,

1

ACCEPTED MANUSCRIPT

1

4

H, 4CH), 7.75 (d, 2H, 2’,6’CH, J = 7.27 Hz), 7.82 (d, 1H, 7CH, J = 8.4 Hz), 9.02 (brs, 1H,

1

3

”NH, D O exchangeable), 12.26 (brs, 1H, 2”NH, D O exchangeable); CNMR δ (ppm)

2

(

DMSO-d

6

3 2 3

, TMS): 14.6 (SCH ), 35.5 (CH ), 55.6 (OCH ), 66.2 (5”CH), 101.4 (C-4), 109.8 (C-

3

), 110.3 (C-7), 112.2 (C-6), 114.8 (CH=CH ), 124.1 (2’,6’CH), 127.3 (4’CH), 127.8 (CH-C (q)

2

fused carbon), 128.5 (3’,5’CH), 130.3 (C-2), 133.6 (1’CH), 134.2 (C-N (q) fused carbon), 135.5

CH=CH ), 149.0 (C-5), 160.8 (6”CH), 185.0 (C=S); MS (70 ev): m/z = 392.12 (M+1); Anal.

(

2

Calcd. for C H N OS (391.49): C, 64.43; H, 5.41; N, 17.89, Found, C, 64.50; H, 5.38; N,

2

1

21

5

1

7.93.

4

.1.5.6.

6-(3-allyl-5-(methylthio)-2-phenyl-1H-indol-1-yl)-5-amino-4,5-dihydro-1,2,4-

triazine-3(2H)-thione (5f)

Yield: 78%; m.p. 207-211 ºC; IR (KBr) υ (cm ): 3092 (C-H Ar str), 2950 (C-H str), 1624 (N-

C=N str), 1605, 1470 (C=C str), 1550 (Het. Triazine str), 1527 (C=C Ar str); HNMR (300 MHz,

-1

1

DMSO-d

6 2 3 2

) δ: 2.10 (m, 1H, CH=CH , J = 1.7 Hz), 2.35 (s, 3H, SCH ), 3.14 (d, 2H, CH , J = 6.7

Hz), 4.00 (s, 1H, 5”CH), 4.17 (brs, 2H, 5”NH , D O exchangeable), 5.08 (d, 1H, H =CH , J =

2

A

2

1

.7 Hz), 5.31 (dd, 1H, H

Hz, J = 8.0 Hz), 7.28-7.41 (m, 3H, 3’,4’,5’CH, J = 4.6 Hz, J = 8.6 Hz), 7.66 (s, 1H, 4CH), 7.72

2

d, 2H, 2’,6’CH, J = 7.29 Hz), 7.79 (d, 1H, 7CH, J = 8.0 Hz), 9.04 (brs, 1H, 4”NH, D O

B 2

=CH , J = 0.8 Hz, J = 1.8 Hz, J = 6.7 Hz), 7.10 (dd, 1H, 6CH, J = 2.0

(

1

3

exchangeable), 12.22 (brs, 1H, 2”NH, D O exchangeable); CNMR δ (ppm) (DMSO-d , TMS):

2

6

3

1

5.2 (CH

2

), 66.6 (5”CH), 109.3 (C-3), 111.4 (C-7), 112.0 (C-6), 114.6 (CH=CH

2

), 119.5 (C-4),

24.4 (2’,6’CH), 127.2 (4’CH), 127.7 (CH-C (q) fused carbon), 128.4 (3’,5’CH), 130.6 (C-2),

32.1 (C-5), 133.3 (1’CH), 134.4 (C-N (q) fused carbon), 135.0 (CH=CH ), 161.2 (6”CH), 185.5

2

(C=S); MS (70 ev): m/z = 408.10 (M+1); MS (70 ev): m/z = 408.23 (M+1); Anal. Calcd. for

21 21 5 2

C H N S

(407.55): C, 61.89; H, 5.19; N, 17.18, Found, C, 61.83; H, 5.22; N, 17.23.

4

.1.6. General procedure for the preparation of compound (6a-f)

To a stirring solution of indoles 4a-f (10 mmol) in dichloromethane (20 mL) in inert condition at

°C was added SnCl (12 mmol) in a single portion via syringe. After the ice bath was removed,

0

4

the mixture was stirred at room temperature for 30 min, and then acetic anhydride (10 mmol)

was added in small portions to the suspension, followed by nitromethane (15 mL). The mixture

was stirred for 2 h at room temperature. After being quenched with ice and water (30 mL), the

mixture was filtered to remove inorganic precipitates, and the organic material was extracted

with ethyl acetate (50 mL). The organic phase was dried over anhydrous sodium sulphate and

concentrated at reduced pressure to give the product as a crystalline solid.

4

.1.6.1.

1-(1-(5-amino-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)-5-chloro-2-phenyl-

H-indol-3-yl)ethanone (6a)

1

-1

Yield: 71%; m.p. 205-208 ºC; IR (KBr) υ (cm ): 3091 (C-H Ar str), 2948 (C-H str), 1682 (C=O

str), 1627 (N-C=N str), 1547 (Het. Triazine str), 1529 (C=C Ar str), 1054 (C-Cl str); HNMR

1

(

300 MHz, DMSO-d ) δ: 2.65 (s, 3H, COCH ), 3.95 (s, 1H, 5”CH), 4.20 (brs, 2H, 5”NH , D O

6 3 2 2

1

2

ACCEPTED MANUSCRIPT

exchangeable), 7.07 (dd, 1H, 6CH, J = 2.5 Hz, J = 8.2 Hz), 7.25-7.40 (m, 3H, 3’,4’,5’CH, J = 4.8

Hz, J = 8.7 Hz), 7.70 (d, 2H, 2’,6’CH, J = 7.30 Hz), 7.75 (d, 1H, 7CH, J = 8.2 Hz), 8.24 (s, 1H,

4

CH), 9.02 (brs, 1H, 4”NH, D

2

O exchangeable), 12.25 (brs, 1H, 2”NH, D

2

O exchangeable);

1

3

CNMR δ (ppm) (DMSO-d , TMS): 28.5 (CH ), 66.6 (5”CH), 109.7 (C-3), 110.5 (C-7), 119.1

6

3

(

1

C-4), 121.4 (C-6), 124.2 (2’,6’CH), 126.3 (C-5), 127.2 (4’CH), 127.6 (CH-C (q) fused carbon),

28.4 (3’,5’CH), 130.9 (C-2), 133.5 (1’CH), 134.2 (C-N (q) fused carbon), 161.1 (6”CH), 185.0

C=S), 199.9 (C=O); MS (70 ev): m/z = 398.45 (M+1); Anal. Calcd. for C H ClN OS

(

1

9

16

5

397.88): C, 57.35; H, 4.05; N, 17.60, Found, C, 57.40; H, 4.12; N, 17.55.

4

.1.6.2.

1-(1-(5-amino-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)-5-fluoro-2-phenyl-

H-indol-3-yl)ethanone (6b)

1

-1

Yield: 73%; m.p. 210-214 ºC; IR (KBr) υ (cm ): 3098 (C-H Ar str), 2954 (C-H str), 1690 (C=O

str), 1632 (N-C=N str), 1549 (Het. Triazine str), 1535 (C=C Ar str), 1178 (C-F str); HNMR (300

1

6 3 2 2

MHz, DMSO-d ) δ: 2.63 (s, 3H, COCH ), 3.96 (s, 1H, 5”CH), 4.22 (brs, 2H, 5”NH , D O

exchangeable), 7.10 (dd, 1H, 6CH, J = 2.2 Hz, J = 8.4 Hz), 7.23-7.35 (m, 3H, 3’,4’,5’CH, J = 4.8

Hz, J = 8.9 Hz), 7.68 (d, 2H, 2’,6’CH, J = 7.31 Hz), 7.85 (d, 1H, 7CH, J = 8.3 Hz), 8.43 (s, 1H,

4

CH), 9.00 (brs, 1H, 4”NH, D O exchangeable), 12.22 (brs, 1H, 2”NH, D O exchangeable);

2

1

3

CNMR δ (ppm) (DMSO-d

6

, TMS): 28.2 (CH

3

), 66.3 (5”CH), 110.0 (C-3), 110.8 (C-7), 111.5

(

C-4), 117.1 (C-6), 124.5 (2’,6’CH), 127.2 (4’CH), 127.6 (CH-C (q) fused carbon), 128.1

3’,5’CH), 130.4 (C-2), 133.0 (1’CH), 134.4 (C-N (q) fused carbon), 160.6 (6”CH), 161.3 (C-5),

1

85.0 (C=S), 200.2 (C=O); MS (70 ev): m/z = 382.19 (M+1); Anal. Calcd. for C H FN OS

19 16 5

(381.43): C, 59.83; H, 4.23; N, 18.36, Found, C, 59.80; H, 4.29; N, 18.39.

4

.1.6.3.

1-(1-(5-amino-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)-5-nitro-2-phenyl-

H-indol-3-yl)ethanone (6c)

1

-1

Yield: 79%; m.p. 219-221 ºC; IR (KBr) υ (cm ): 3097 (C-H Ar str), 2958 (C-H str), 1688 (C=O

str), 1650 (N-C=N str), 1555 (Het. Triazine str), 1530 (C=C Ar str), 1345 (NO str); HNMR

1

2

(

300 MHz, DMSO-d ) δ: 2.62 (s, 3H, COCH ), 3.95 (s, 1H, 5”CH), 4.21 (brs, 2H, 5”NH , D O

6 3 2 2

exchangeable), 7.17 (dd, 1H, 6CH, J = 2.2 Hz, J = 8.1 Hz), 7.25-7.37 (m, 3H, 3’,4’,5’CH, J = 4.9

Hz, J = 8.7 Hz), 7.72 (d, 2H, 2’,6’CH, J = 7.30 Hz), 7.93 (d, 1H, 7CH, J = 8.1 Hz), 8.69 (s, 1H,

4

CH), 9.05 (brs, 1H, 4”NH, D

2

O exchangeable), 12.24 (brs, 1H, 2”NH, D

2

O exchangeable);

1

3

CNMR δ (ppm) (DMSO-d , TMS): 28.3 (CH ), 66.5 (5”CH), 108.8 (C-7), 110.2 (C-3), 116.6

6

3

(

C-6), 124.7 (2’,6’CH), 127.1 (4’CH), 127.5 (C-4), 127.9 (CH-C (q) fused carbon), 128.4

3’,5’CH), 130.5 (C-2), 132.8 (C-5), 133.8 (1’CH), 140.4 (C-N (q) fused carbon), 160.9 (6”CH),

1

85.4 (C=S), 200.0 (C=O); MS (70 ev): m/z = 409.05 (M+1); Anal. Calcd. for C19

16 6 3

H N O S

(408.43): C, 55.87; H, 3.95; N, 20.58, Found, C, 55.93; H, 3.90; N, 20.50.

4

.1.6.4.

1-(1-(5-amino-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)-2-phenyl-5-

(trifluoromethyl)-1H-indol-3-yl)ethanone (6d)

1

3

ACCEPTED MANUSCRIPT

-1

Yield: 72%; m.p. 225-228 ºC; IR (KBr) υ (cm ): 3090 (C-H Ar str), 2950 (C-H str), 1685 (C=O

1

str), 1631 (N-C=N str), 1551 (Het. Triazine str), 1525 (C=C Ar str), 1190 (C-F str); HNMR (300

6 3 2 2

MHz, DMSO-d ) δ: 2.60 (s, 3H, COCH ), 3.93 (s, 1H, 5”CH), 4.22 (brs, 2H, 5”NH , D O

exchangeable), 7.22-7.38 (m, 3H, 3’,4’,5’CH, J = 4.7 Hz, J = 8.5 Hz), 7.50 (dd, 1H, 6CH, J = 2.0

Hz, J = 7.8 Hz), 7.70 (d, 2H, 2’,6’CH, J = 7.26 Hz), 7.85 (d, 1H, 7CH, J = 8.0 Hz), 8.84 (s, 1H,

4

CH), 9.01 (brs, 1H, 4”NH, D O exchangeable), 12.25 (brs, 1H, 2”NH, D O exchangeable);

2 2

1

3

CNMR δ (ppm) (DMSO-d , TMS): 28.6 (CH ), 66.5 (5”CH), 110.2 (C-3), 111.7 (C-7), 114.9

6

3

(

C-6), 117.5 (C-4), 124.1 (2’,6’CH), 124.6 (CF ), 125.4 (C-5), 127.5 (4’CH), 128.0 (CH-C (q)

3

fused carbon), 128.6 (3’,5’CH), 130.5 (C-2), 133.1 (1’CH), 134.8 (C-N (q) fused carbon), 161.2

6”CH), 185.3 (C=S), 200.5 (C=O); MS (70 ev): m/z = 432.39 (M+1); Anal. Calcd. for

C H F N OS (431.43): C, 55.68; H, 3.74; N, 16.23, Found, C, 55.73; H, 3.68; N, 16.29.

(

20

16

3

5

4

.1.6.5.

1-(1-(5-amino-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)-5-methoxy-2-

phenyl-1H-indol-3-yl)ethanone (6e)

Yield: 77%; m.p. 198-201 ºC; IR (KBr) υ (cm ): 3095 (C-H Ar str), 2952 (C-H str), 1686 (C=O

str), 1634 (N-C=N str), 1549 (Het. Triazine str), 1531 (C=C Ar str), 1248 (C-O-C str); HNMR

-1

1

(300 MHz, DMSO-d ) δ: 2.66 (s, 3H, COCH ), 3.70 (s, 3H, OCH ), 3.98 (s, 1H, 5”CH), 4.20

6 3 3

(

3

7

brs, 2H, 5”NH

2

, D

H, 3’,4’,5’CH, J = 4.8 Hz, J = 8.6 Hz), 7.65 (s, 1H, 4CH), 7.70 (d, 2H, 2’,6’CH, J = 7.27 Hz),

.80 (d, 1H, 7CH, J = 8.4 Hz), 9.02 (brs, 1H, 4”NH, D O exchangeable), 12.25 (brs, 1H, 2”NH,

2

O exchangeable), 7.09 (dd, 1H, 6CH, J = 2.0 Hz, J = 8.2 Hz), 7.26-7.39 (m,

2

1

3

D O exchangeable); CNMR δ (ppm) (DMSO-d , TMS): 28.4 (CH ), 55.5 (OCH ), 66.9

2

6

3

(

5”CH), 101.5 (C-4), 110.1 (C-3), 110.5 (C-7), 112.4 (C-6), 124.1 (2’,6’CH), 127.2 (4’CH),

27.6 (CH-C (q) fused carbon), 128.4 (3’,5’CH), 130.3 (C-2), 133.5 (1’CH), 134.0 (C-N (q)

fused carbon), 149.1 (C-5), 160.6 (6”CH), 185.0 (C=S), 200.4 (C=O); MS (70 ev): m/z = 394.27

1

(

M+1); Anal. Calcd. for C H N O S (393.46): C, 61.05; H, 4.87; N, 17.80, Found, C, 61.12; H,

20 19 5 2

4

.81; N, 17.85.

4

.1.6.6.

1-(1-(5-amino-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)-5-(methylthio)-2-

phenyl-1H-indol-3-yl)ethanone (6f)

Yield: 82%; m.p. 192-195 ºC; IR (KBr) υ (cm ): 3098 (C-H Ar str), 2958 (C-H str), 1690 (C=O

str), 1625 (N-C=N str), 1546 (Het. Triazine str), 1533 (C=C Ar str); HNMR (300 MHz, DMSO-

-1

1

d ) δ: 2.40 (s, 3H, COCH ), 2.62 (s, 3H, SCH ), 3.97 (s, 1H, 5”CH), 4.24 (brs, 2H, 5”NH , D O

6

3

2

exchangeable), 7.27-7.42 (m, 3H, 3’,4’,5’CH, J = 4.5 Hz, J = 8.2 Hz), 7.45 (dd, 1H, 6CH, J = 1.9

Hz, J = 7.9 Hz), 7.69 (d, 2H, 2’,6’CH, J = 7.29 Hz), 7.85 (d, 1H, 7CH, J = 8.1 Hz), 7.90 (s, 1H,

4

CH), 9.05 (brs, 1H, 4”NH, D

2

O exchangeable), 12.20 (brs, 1H, 2”NH, D

2

O exchangeable);

1

3

CNMR δ (ppm) (DMSO-d , TMS): 15.2 (SCH ), 28.8 (CH ), 66.4 (5”CH), 110.1 (C-3), 111.5

6

3

(

1

(

C-7), 112.2 (C-6), 119.4 (C-4), 124.5 (2’,6’CH), 127.1 (4’CH), 127.8 (CH-C (q) fused carbon),

28.2 (3’,5’CH), 130.9 (C-2), 132.5 (C-5), 133.1 (1’CH), 134.0 (C-N (q) fused carbon), 161.0

6”CH), 185.2 (C=S), 200.7 (C=O); MS (70 ev): m/z = 410.25 (M+1); Anal. Calcd. for

C H N OS (409.53): C, 58.66; H, 4.68; N, 17.10, Found, C, 58.71; H, 4.72; N, 17.05.

20

19

5

2

1

4

ACCEPTED MANUSCRIPT

4

.1.7. General procedure for the preparation of compound (7a-f)

The indole-triazine derivatives 4a-f was N-protected by 9-fluorenylmethoxycarbonyl (Fmoc) and

converted smoothly to sulfonyl chloride via treatment with sulfuryl chloride in

dimethylformamide and refluxed for 6 h. Sulfonyl chloride was then reacted with ammonia in a

parallel fashion to generate sulfonamide represented by general structure 7a-f. Finally,

concomitant deprotection of the Fmoc group at the triazine N-2 and N-4 was executed after

completion of the reaction by adding 4-(aminomethyl)piperidine (0.5-2 mL) with continuous

stirring to provide the sulfonamide derivative that was purified by crystallization in petroleum

ether: dichloromethane (4:1) to give pure compounds 7a-f.

4

.1.7.1.

1-(5-amino-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)-5-chloro-2-phenyl-

H-indole-3-sulfonamide (7a)

1

-1

Yield: 80%; m.p. 172-175 ºC; IR (KBr) υ (cm ): 3360 (N-H str, br), 3096 (C-H Ar str), 1634 (N-

C=N str), 1545 (Het. Triazine str), 1533 (C=C Ar str), 1326, 1143 (S=O str), 1054 (C-Cl str);

1

HNMR (300 MHz, DMSO-d

.05 (dd, 1H, 6CH, J = 2.4 Hz, J = 8.2 Hz), 7.25-7.38 (m, 3H, 3’,4’,5’CH, J = 4.6 Hz, J = 8.5

Hz), 7.65 (brs, 2H, SO NH , D O exchangeable), 7.72 (d, 2H, 2’,6’CH, J = 7.31 Hz), 7.77 (d,

6 2 2

) δ: 3.97 (s, 1H, 5”CH), 4.20 (brs, 2H, 5”NH , D O exchangeable),

7

2

1

H, 7CH, J = 8.1 Hz), 8.34 (s, 1H, 4CH), 9.04 (brs, 1H, 4”NH, D O exchangeable), 12.21 (brs,

2

1

3

H, 2”NH, D

2

O exchangeable); CNMR δ (ppm) (DMSO-d

6

, TMS): 66.5 (5”CH), 110.2 (C-7),

14.8 (C-3), 119.5 (C-4), 120.9 (C-6), 124.0 (2’,6’CH), 126.4 (C-5), 127.1 (4’CH), 127.5 (CH-C

(

1

q) fused carbon), 128.5 (3’,5’CH), 130.7 (C-2), 133.2 (1’CH), 134.7 (C-N (q) fused carbon),

61.4 (6”CH), 185.2 (C=S); MS (70 ev): m/z = 435.70 (M+1); Anal. Calcd. for C H ClN O S

2 2

17

15

6

(434.92): C, 46.95; H, 3.48; N, 19.32, Found, C, 47.00; H, 3.42; N, 19.27.

4

.1.7.2.

1-(5-amino-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)-5-fluoro-2-phenyl-1H-

indole-3-sulfonamide (7b)

Yield: 86%; m.p. 177-179 ºC; IR (KBr) υ (cm ): 3369 (N-H str, br), 3090 (C-H Ar str), 1631 (N-

-1

C=N str), 1550 (Het. Triazine str), 1528 (C=C Ar str), 1328, 1140 (S=O str), 1170 (C-F str);

1

HNMR (300 MHz, DMSO-d ) δ: 3.94 (s, 1H, 5”CH), 4.21 (brs, 2H, 5”NH , D O exchangeable),

6

2

7

.09 (dd, 1H, 6CH, J = 2.2 Hz, J = 8.3 Hz), 7.24-7.37 (m, 3H, 3’,4’,5’CH, J = 4.5 Hz, J = 8.8

Hz), 7.60 (brs, 2H, SO NH , D O exchangeable), 7.71 (d, 2H, 2’,6’CH, J = 7.30 Hz), 7.83 (d,

2

1

H, 7CH, J = 8.4 Hz), 8.47 (s, 1H, 4CH), 9.01 (brs, 1H, 4”NH, D

2

O exchangeable), 12.19 (brs,

1

3

H, 2”NH, D O exchangeable); CNMR δ (ppm) (DMSO-d , TMS): 66.5 (5”CH), 110.8 (C-7),

2

6

11.4 (C-4), 114.5 (C-3), 117.5 (C-6), 124.3 (2’,6’CH), 127.1 (4’CH), 127.8 (CH-C (q) fused

carbon), 128.2 (3’,5’CH), 130.2 (C-2), 133.5 (1’CH), 134.5 (C-N (q) fused carbon), 160.5

6”CH), 161.3 (C-5), 184.9 (C=S); MS (70 ev): m/z = 419.11 (M+1); Anal. Calcd. for

C H FN O S (418.47): C, 48.79; H, 3.61; N, 20.08, Found, C, 48.85; H, 3.57; N, 20.01.

(

17

15

6

2 2

1

5

ACCEPTED MANUSCRIPT

4

.1.7.3.

1-(5-amino-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)-5-nitro-2-phenyl-1H-

indole-3-sulfonamide (7c)

Yield: 70%; m.p. 215-218 ºC; IR (KBr) υ (cm ): 3373 (N-H str, br), 3099 (C-H Ar str), 1630 (N-

-1

C=N str), 1552 (Het. Triazine str), 1530 (C=C Ar str), 1340 (NO str), 1325, 1140 (S=O str);

2

1

HNMR (300 MHz, DMSO-d ) δ: 3.96 (s, 1H, 5”CH), 4.20 (brs, 2H, 5”NH , D O exchangeable),

6

2

7

.18 (dd, 1H, 6CH, J = 2.2 Hz, J = 8.1 Hz), 7.25-7.34 (m, 3H, 3’,4’,5’CH, J = 4.8 Hz, J = 8.7

Hz), 7.75 (d, 2H, 2’,6’CH, J = 7.28 Hz), 7.84 (brs, 2H, SO NH , D O exchangeable), 7.92 (d,

2

1

H, 7CH, J = 8.2 Hz), 8.71 (s, 1H, 4CH), 9.05 (brs, 1H, 4”NH, D O exchangeable), 12.23 (brs,

2

1

3

H, 2”NH, D O exchangeable); CNMR δ (ppm) (DMSO-d , TMS): 66.2 (5”CH), 108.3 (C-7),

2

6

14.5 (C-3), 116.4 (C-6), 124.3 (2’,6’CH), 126.9 (4’CH), 127.2 (C-4), 127.6 (CH-C (q) fused

carbon), 128.6 (3’,5’CH), 130.4 (C-2), 132.3 (C-5), 133.8 (1’CH), 140.4 (C-N (q) fused carbon),

61.2 (6”CH), 185.1 (C=S); MS (70 ev): m/z = 446.33 (M+1); Anal. Calcd. for C17

445.48): C, 45.83; H, 3.39; N, 22.01, Found, C, 45.90; H, 3.32; N, 21.95.

1

15 7 4 2

H N O S

(

4

.1.7.4.

1-(5-amino-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)-2-phenyl-5-

trifluoromethyl)-1H-indole-3-sulfonamide (7d)

(

-1

Yield: 73%; m.p. 191-194 ºC; IR (KBr) υ (cm ): 3374 (N-H str, br), 3093 (C-H Ar str), 1625 (N-

C=N str), 1554 (Het. Triazine str), 1532 (C=C Ar str), 1327, 1142 (S=O str), 1199 (C-F str);

1

HNMR (300 MHz, DMSO-d ) δ: 3.94 (s, 1H, 5”CH), 4.23 (brs, 2H, 5”NH , D O exchangeable),

6

2

7

.22-7.38 (m, 3H, 3’,4’,5’CH, J = 4.7 Hz, J = 8.5 Hz), 7.52 (dd, 1H, 6CH, J = 2.0 Hz, J = 8.1

Hz), 7.71 (d, 2H, 2’,6’CH, J = 7.26 Hz), 7.80 (brs, 2H, SO NH , D O exchangeable), 7.88 (d,

2

1

H, 7CH, J = 8.2 Hz), 8.85 (s, 1H, 4CH), 9.03 (brs, 1H, 4”NH, D

2

O exchangeable), 12.20 (brs,

1

3

H, 2”NH, D O exchangeable); CNMR δ (ppm) (DMSO-d , TMS): 66.6 (5”CH), 111.5 (C-7),

2

6

3

14.7 (C-3), 115.3 (C-6), 117.2 (C-4), 124.0 (2’,6’CH), 124.5 (CF ), 125.3 (C-5), 127.6 (4’CH),

28.2 (CH-C (q) fused carbon), 128.4 (3’,5’CH), 130.3 (C-2), 133.0 (1’CH), 134.7 (C-N (q)

fused carbon), 161.2 (6”CH), 185.3 (C=S); MS (70 ev): m/z = 469.34 (M+1); Anal. Calcd. for

C H F N O S (468.48): C, 46.15; H, 3.23; N, 17.94, Found, C, 46.20; H, 3.20; N, 17.89.

18

15

3

6

2 2

4

.1.7.5.

1-(5-amino-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)-5-methoxy-2-phenyl-

H-indole-3-sulfonamide (7e)

1

-1

Yield: 81%; m.p. 182-184 ºC; IR (KBr) υ (cm ): 3365 (N-H str, br), 3088 (C-H Ar str), 2958 (C-

H str), 1636 (N-C=N str), 1557 (Het. Triazine str), 1528 (C=C Ar str), 1332, 1141 (S=O str),

1

4

250 (C-O-C str); HNMR (300 MHz, DMSO-d

6

) δ: 3.72 (s, 3H, OCH

3

), 3.99 (s, 1H, 5”CH),

.20 (brs, 2H, 5”NH , D O exchangeable), 7.11 (dd, 1H, 6CH, J = 2.1 Hz, J = 8.3 Hz), 7.26-7.41

2

(m, 3H, 3’,4’,5’CH, J = 4.8 Hz, J = 8.4 Hz), 7.68 (s, 1H, 4CH), 7.74 (d, 2H, 2’,6’CH, J = 7.27

Hz), 7.69 (brs, 2H, SO NH , D O exchangeable), 7.81 (d, 1H, 7CH, J = 8.4 Hz), 9.02 (brs, 1H,

2

1

3

4

”NH, D

2 2

O exchangeable), 12.24 (brs, 1H, 2”NH, D O exchangeable); CNMR δ (ppm)

(

DMSO-d , TMS): 55.7 (OCH ), 66.5 (5”CH), 101.6 (C-4), 110.5 (C-7), 112.3 (C-6), 114.6 (C-

6

3

1

), 124.0 (2’,6’CH), 127.4 (4’CH), 127.5 (CH-C (q) fused carbon), 128.5 (3’,5’CH), 130.4 (C-2),

33.5 (1’CH), 134.3 (C-N (q) fused carbon), 149.2 (C-5), 160.8 (6”CH), 185.1 (C=S); MS (70

1

6

ACCEPTED MANUSCRIPT

ev): m/z = 431.09 (M+1); Anal. Calcd. for C18

Found, C, 50.28; H, 4.17; N, 19.47.

18 6 3 2

H N O S (430.50): C, 50.22; H, 4.21; N, 19.52,

4

.1.7.6.

1-(5-amino-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)-5-(methylthio)-2-

phenyl-1H-indole-3-sulfonamide (7f)

Yield: 77%; m.p. 217-220 ºC; IR (KBr) υ (cm ): 3372 (N-H str, br), 3095 (C-H Ar str), 2960 (C-

-1

H str), 1626 (N-C=N str), 1554 (Het. Triazine str), 1529 (C=C Ar str), 1325, 1145 (S=O str);

1

HNMR (300 MHz, DMSO-d ) δ: 2.38 (s, 3H, SCH ), 3.95 (s, 1H, 5”CH), 4.22 (brs, 2H, 5”NH ,

6

3

2

D O exchangeable), 7.25-7.40 (m, 3H, 3’,4’,5’CH, J = 4.5 Hz, J = 8.2 Hz), 7.43 (dd, 1H, 6CH, J

2

=

2.0 Hz, J = 7.9 Hz), 7.61 (brs, 2H, SO NH , D O exchangeable), 7.72 (d, 2H, 2’,6’CH, J =

2 2 2

7

.28 Hz), 7.83 (d, 1H, 7CH, J = 8.1 Hz), 7.95 (s, 1H, 4CH), 9.05 (brs, 1H, 4”NH, D O

2

1

3

2 6

exchangeable), 12.26 (brs, 1H, 2”NH, D O exchangeable); CNMR δ (ppm) (DMSO-d , TMS):

1

6.0 (SCH ), 66.7 (5”CH), 111.3 (C-7), 112.3 (C-6), 114.8 (C-3), 119.3 (C-4), 124.5 (2’,6’CH),

27.0 (4’CH), 127.6 (CH-C (q) fused carbon), 128.4 (3’,5’CH), 130.7 (C-2), 132.2 (C-5), 133.5

3

(

1’CH), 134.5 (C-N (q) fused carbon), 161.0 (6”CH), 185.0 (C=S); MS (70 ev): m/z = 447.26

M+1); Anal. Calcd. for C18 (446.57): C, 48.41; H, 4.06; N, 18.82, Found, C, 48.45;

18 6 2 3

H N O S

H, 4.01; N, 18.75.

4

.1.8. General procedure for the preparation of compound (8a-f)

To a solution of compounds 4a-f (22.3 mmol) in anhydrous N,N-dimethylformamide (10 mL),

trichloroacetic acid anhydride (33.5 mmol) was added and the mixture was stirred for 6 hours at

0

°C. The saturated sodium bicarbonate solution was added to the mixture and the resultant

mixture was extracted with ethyl acetate. The organic layer was washed with water and saturated

sodium chloride solution, dried over anhydrous sodium sulfate and then concentrated in vacuum.

To a solution of the residue in tetrahydrofuran (135 mL), 4M sodium hydroxide solution (65.2

mmol) was added and stirred at room temperature for 6 hours. The mixture was diluted with

diethyl ether and 1M sodium hydroxide solution. The aqueous layer was washed with diethyl

ether and neutralized with 1M hydrochloric acid. The precipitate was separated from the

resultant aqueous solution and dried in vacuum to give compounds 8a-f.

4

.1.8.1.

1-(5-amino-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)-5-chloro-2-phenyl-

1

H-indole-3-carboxylic acid (8a)

-1

Yield: 85%; m.p. 164-168 ºC; IR (KBr) υ (cm ): 3350 (OH str, br), 3092 (C-H Ar str), 1715

C=O str), 1627 (N-C=N str), 1555 (Het. Triazine str), 1530 (C=C Ar str), 1055 (C-Cl str);

HNMR (300 MHz, DMSO-d

.05 (dd, 1H, 6CH, J = 1.9 Hz, J = 8.4 Hz), 7.21-7.37 (m, 3H, 3’,4’,5’CH, J = 4.8 Hz, J = 8.5

Hz), 7.72 (d, 2H, 2’,6’CH, J = 7.29 Hz), 7.78 (d, 1H, 7CH, J = 8.3 Hz), 8.21 (s, 1H, 4CH), 9.06

(

1

6 2 2

) δ: 3.90 (s, 1H, 5”CH), 4.25 (brs, 2H, 5”NH , D O exchangeable),

7

(

brs, 1H, 4”NH, D O exchangeable), 11.05 (brs, 1H, COOH), 12.24 (brs, 1H, 2”NH, D O

2

1

3

exchangeable); CNMR δ (ppm) (DMSO-d

6

, TMS): 66.5 (5”CH), 110.2 (C-7), 110.6 (C-3),

1

19.0 (C-4), 121.2 (C-6), 124.2 (2’,6’CH), 126.0 (C-5), 127.1 (4’CH), 127.6 (CH-C (q) fused

1

7

ACCEPTED MANUSCRIPT

carbon), 128.5 (3’,5’CH), 130.8 (C-2), 133.5 (1’CH), 134.8 (C-N (q) fused carbon), 161.0

(6”CH), 170.5 (C=O), 185.5 (C=S); MS (70 ev): m/z = 400.65 (M+1); Anal. Calcd. for

C

18

H

14ClN

5

O

2

S (399.85): C, 54.07; H, 3.53; N, 17.51, Found, C, 54.13; H, 3.47; N, 17.56.

4

.1.8.2.

1-(5-amino-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)-5-fluoro-2-phenyl-

1

H-indole-3-carboxylic acid (8b)

-1

Yield: 90%; m.p. 172-174 ºC; IR (KBr) υ (cm ): 3356 (OH str, br), 3091 (C-H Ar str), 1718

(

C=O str), 1625 (N-C=N str), 1558 (Het. Triazine str), 1535 (C=C Ar str), 1168 (C-F str);

1

HNMR (300 MHz, DMSO-d ) δ: 3.92 (s, 1H, 5”CH), 4.23 (brs, 2H, 5”NH , D O exchangeable),

6

2

7

.08 (dd, 1H, 6CH, J = 2.2 Hz, J = 8.3 Hz), 7.23-7.38 (m, 3H, 3’,4’,5’CH, J = 4.8 Hz, J = 8.7

Hz), 7.68 (d, 2H, 2’,6’CH, J = 7.33 Hz), 7.85 (d, 1H, 7CH, J = 8.5 Hz), 8.44 (s, 1H, 4CH), 9.02

brs, 1H, 4”NH, D O exchangeable), 11.02 (brs, 1H, COOH), 12.25 (brs, 1H, 2”NH, D

exchangeable); CNMR δ (ppm) (DMSO-d , TMS): 66.1 (5”CH), 110.3 (C-3), 110.9 (C-7),

(

2

O

1

3

6

1

(

1

(

11.5 (C-4), 117.0 (C-6), 124.6 (2’,6’CH), 127.2 (4’CH), 127.6 (CH-C (q) fused carbon), 128.4

3’,5’CH), 130.1 (C-2), 133.0 (1’CH), 134.4 (C-N (q) fused carbon), 160.6 (6”CH), 161.4 (C-5),

70.8 (C=O), 185.1 (C=S); MS (70 ev): m/z = 384.02 (M+1); Anal. Calcd. for C18

383.40): C, 56.39; H, 3.68; N, 18.27, Found, C, 56.45; H, 3.72; N, 18.21.

5 2

H14FN O S

4

.1.8.3.

indole-3-carboxylic acid (8c)

Yield: 83%; m.p. 181-183 ºC; IR (KBr) υ (cm ): 3351 (OH str, br), 3098 (C-H Ar str), 1709

1-(5-amino-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)-5-nitro-2-phenyl-1H-

-1

(

C=O str), 1634 (N-C=N str), 1547 (Het. Triazine str), 1528 (C=C Ar str), 1335 (NO

2

str);

1

HNMR (300 MHz, DMSO-d ) δ: 3.95 (s, 1H, 5”CH), 4.20 (brs, 2H, 5”NH , D O exchangeable),

6

2

7

.18 (dd, 1H, 6CH, J = 2.0 Hz, J = 8.1 Hz), 7.25-7.35 (m, 3H, 3’,4’,5’CH, J = 4.9 Hz, J = 8.5

Hz), 7.74 (d, 2H, 2’,6’CH, J = 7.30 Hz), 7.93 (d, 1H, 7CH, J = 8.2 Hz), 8.68 (s, 1H, 4CH), 9.04

brs, 1H, 4”NH, D O exchangeable), 11.04 (brs, 1H, COOH), 12.23 (brs, 1H, 2”NH, D O

(

2

1

3

exchangeable); CNMR δ (ppm) (DMSO-d , TMS): 66.3 (5”CH), 108.6 (C-7), 110.2 (C-3),

6

1

(

1

16.8 (C-6), 124.3 (2’,6’CH), 127.0 (4’CH), 127.5 (C-4), 127.8 (CH-C (q) fused carbon), 128.5

3’,5’CH), 130.8 (C-2), 132.6 (C-5), 133.8 (1’CH), 140.3 (C-N (q) fused carbon), 161.0 (6”CH),

70.6 (C=O), 185.2 (C=S); MS (70 ev): m/z = 411.17 (M+1); Anal. Calcd. for C H N O S

1

8

14

6

4

(410.41): C, 52.68; H, 3.44; N, 20.48, Found, C, 52.72; H, 3.37; N, 20.40.

4

.1.8.4.

1-(5-amino-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)-2-phenyl-5-

(

trifluoromethyl)-1H-indole-3-carboxylic acid (8d)

-1

Yield: 78%; m.p. 188-190 ºC; IR (KBr) υ (cm ): 3358 (OH str, br), 3090 (C-H Ar str), 1718

C=O str), 1625 (N-C=N str), 1548 (Het. Triazine str), 1530 (C=C Ar str), 1209 (C-F str);

HNMR (300 MHz, DMSO-d

.22-7.36 (m, 3H, 3’,4’,5’CH, J = 4.7 Hz, J = 8.2 Hz), 7.53 (dd, 1H, 6CH, J = 2.0 Hz, J = 8.0

Hz), 7.75 (d, 2H, 2’,6’CH, J = 7.25 Hz), 7.88 (d, 1H, 7CH, J = 8.1 Hz), 8.87 (s, 1H, 4CH), 9.04

brs, 1H, 4”NH, D O exchangeable), 11.02 (brs, 1H, COOH), 12.25 (brs, 1H, 2”NH, D O

(

1

6 2 2

) δ: 3.91 (s, 1H, 5”CH), 4.22 (brs, 2H, 5”NH , D O exchangeable),

7

(

2

1

8

ACCEPTED MANUSCRIPT

1

3

exchangeable); CNMR δ (ppm) (DMSO-d

6

, TMS): 66.3 (5”CH), 110.5 (C-3), 111.8 (C-7),

1

15.2 (C-6), 117.1 (C-4), 124.1 (2’,6’CH), 124.4 (CF ), 125.7 (C-5), 127.7 (4’CH), 128.1 (CH-C

3

(

1

q) fused carbon), 128.5 (3’,5’CH), 130.2 (C-2), 133.4 (1’CH), 134.7 (C-N (q) fused carbon),

61.0 (6”CH), 170.8 (C=O), 184.9 (C=S); MS (70 ev): m/z = 434.20 (M+1); Anal. Calcd. for

C H F N O S (433.41): C, 52.65; H, 3.26; N, 16.16, Found, C, 52.60; H, 3.22; N, 16.21.

19

14

3

5

2

4

.1.8.5.

1-(5-amino-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)-5-methoxy-2-phenyl-

H-indole-3-carboxylic acid (8e)

1

-1

Yield: 82%; m.p. 166-169 ºC; IR (KBr) υ (cm ): 3360 (OH str, br), 3098 (C-H Ar str), 2965 (C-

H str), 1726 (C=O str), 1625 (N-C=N str), 1560 (Het. Triazine str), 1536 (C=C Ar str), 1255 (C-

1

O-C str); HNMR (300 MHz, DMSO-d ) δ: 3.73 (s, 3H, OCH ), 3.97 (s, 1H, 5”CH), 4.18 (brs,

6

3

2

3

H, 5”NH

’,4’,5’CH, J = 4.8 Hz, J = 8.5 Hz), 7.63 (s, 1H, 4CH), 7.71 (d, 2H, 2’,6’CH, J = 7.27 Hz), 7.81

O exchangeable), 11.05 (brs, 1H, COOH),

2.21 (brs, 1H, 2”NH, D O exchangeable); CNMR δ (ppm) (DMSO-d , TMS): 55.6 (OCH ),

6.7 (5”CH), 101.2 (C-4), 110.4 (C-3), 110.7 (C-7), 112.1 (C-6), 124.3 (2’,6’CH), 127.5 (4’CH),

27.6 (CH-C (q) fused carbon), 128.5 (3’,5’CH), 130.2 (C-2), 133.4 (1’CH), 134.0 (C-N (q)

2 2

, D O exchangeable), 7.08 (dd, 1H, 6CH, J = 2.0 Hz, J = 8.4 Hz), 7.25-7.40 (m, 3H,

(d, 1H, 7CH, J = 8.4 Hz), 9.00 (brs, 1H, 4”NH, D

2

1

3

1

6

1

2

6

3

fused carbon), 149.1 (C-5), 160.6 (6”CH), 170.4 (C=O), 185.0 (C=S); MS (70 ev): m/z = 396.22

(

M+1); Anal. Calcd. for C H N O S (395.43): C, 57.71; H, 4.33; N, 17.71, Found, C, 57.79; H,

19 17 5 3

4

.40; N, 17.66.

4

.1.8.6.

1-(5-amino-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)-5-(methylthio)-2-

phenyl-1H-indole-3-carboxylic acid (8f)

-1

Yield: 87%; m.p. 185-190 ºC; IR (KBr) υ (cm ): 3365 (OH str, br), 3100 (C-H Ar str), 2968 (C-

1

H str), 1730 (C=O str), 1622 (N-C=N str), 1558 (Het. Triazine str), 1532 (C=C Ar str); HNMR

(

300 MHz, DMSO-d ) δ: 2.39 (s, 3H, SCH ), 3.99 (s, 1H, 5”CH), 4.22 (brs, 2H, 5”NH , D O

6 3 2 2

exchangeable), 7.27-7.38 (m, 3H, 3’,4’,5’CH, J = 4.5 Hz, J = 8.2 Hz), 7.44 (dd, 1H, 6CH, J = 1.9

Hz, J = 8.1 Hz), 7.67 (d, 2H, 2’,6’CH, J = 7.29 Hz), 7.82 (d, 1H, 7CH, J = 8.0 Hz), 7.91 (s, 1H,

4

CH), 9.03 (brs, 1H, 4”NH, D O exchangeable), 11.00 (brs, 1H, COOH), 12.22 (brs, 1H, 2”NH,

2

1

3

D O exchangeable); CNMR δ (ppm) (DMSO-d , TMS): 15.5 (SCH ), 66.5 (5”CH), 110.3 (C-

2

6

3

), 111.7 (C-7), 112.2 (C-6), 119.6 (C-4), 124.3 (2’,6’CH), 127.2 (4’CH), 127.6 (CH-C (q) fused

carbon), 128.5 (3’,5’CH), 130.8 (C-2), 132.0 (C-5), 133.3 (1’CH), 134.8 (C-N (q) fused carbon),

61.2 (6”CH), 173.0 (C=O), 185.3 (C=S); MS (70 ev): m/z = 412.28 (M+1); Anal. Calcd. for

C H N O S (411.50): C, 55.46; H, 4.16; N, 17.02, Found, C, 55.50; H, 4.22; N, 16.97.

1

19

17

5

2 2

4

.1.9. General procedure for the preparation of compound (9a-f)

A mixture of carboxylic derivatives 8a-f (0.1 mol), thionyl chloride (10 mL) and dimethyl

formamide (0.1 mL) was refluxed for 12 h. The solution was evaporated and the residue

triturated with toluene (10 mL) and re-evaporated. The resulting solid was stirred at 25 °C with

1

9

ACCEPTED MANUSCRIPT

concentrated aqueous ammonia for 24 h. The carboxamide derivatives 9a-f crystallized from

water as colourless needles.

4

.1.9.1.

1-(5-amino-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)-5-chloro-2-phenyl-

H-indole-3-carboxamide (9a)

Yield: 71%; m.p. 158-161 ºC; IR (KBr) υ (cm ): 3305 (NH str, br), 3092 (C-H Ar str), 1660

1

-1

(

C=O str), 1628 (N-C=N str), 1565 (Het. Triazine str), 1529 (C=C Ar str), 1060 (C-Cl str);

1

HNMR (300 MHz, DMSO-d ) δ: 3.99 (s, 1H, 5”CH), 4.18 (brs, 2H, 5”NH , D O exchangeable),

6

2

4

7

1

.70 (brs, 2H, CONH , D O exchangeable), 7.05 (dd, 1H, 6CH, J = 2.3 Hz, J = 8.5 Hz), 7.22-

2 2

.35 (m, 3H, 3’,4’,5’CH, J = 4.7 Hz, J = 8.5 Hz), 7.72 (d, 2H, 2’,6’CH, J = 7.29 Hz), 7.78 (d,

H, 7CH, J = 8.5 Hz), 8.20 (s, 1H, 4CH), 9.02 (brs, 1H, 4”NH, D O exchangeable), 12.25 (brs,

2

1

3

H, 2”NH, D

2 6

O exchangeable); CNMR δ (ppm) (DMSO-d , TMS): 66.7 (5”CH), 110.0 (C-7),

10.6 (C-3), 119.3 (C-4), 121.1 (C-6), 124.0 (2’,6’CH), 126.3 (C-5), 127.2 (4’CH), 127.7 (CH-C

(q) fused carbon), 128.3 (3’,5’CH), 131.0 (C-2), 133.1 (1’CH), 134.2 (C-N (q) fused carbon),

1

60.7 (6”CH), 172.4 (C=O), 185.3 (C=S); MS (70 ev): m/z = 399.74 (M+1); Anal. Calcd. for

C

18

H

15ClN

6

OS (398.87): C, 54.20; H, 3.79; N, 21.07, Found, C, 54.25; H, 3.72; N, 21.00.

4

.1.9.2.

1-(5-amino-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)-5-fluoro-2-phenyl-

1

H-indole-3-carboxamide (9b)

Yield: 78%; m.p. 173-176 ºC; IR (KBr) υ (cm ): 3309 (NH str, br), 3090 (C-H Ar str), 1665

C=O str), 1633 (N-C=N str), 1562 (Het. Triazine str), 1527 (C=C Ar str), 1135 (C-F str);

-1

(

1

HNMR (300 MHz, DMSO-d

6

) δ: 3.95 (s, 1H, 5”CH), 4.23 (brs, 2H, 5”NH

2 2

, D O exchangeable),

4

7

1

.73 (brs, 2H, CONH , D O exchangeable), 7.07 (dd, 1H, 6CH, J = 2.2 Hz, J = 8.4 Hz), 7.21-

.38 (m, 3H, 3’,4’,5’CH, J = 4.8 Hz, J = 8.7 Hz), 7.68 (d, 2H, 2’,6’CH, J = 7.30 Hz), 7.85 (d,

2

H, 7CH, J = 8.4 Hz), 8.46 (s, 1H, 4CH), 9.04 (brs, 1H, 4”NH, D O exchangeable), 12.22 (brs,

2

1

3

H, 2”NH, D O exchangeable); CNMR δ (ppm) (DMSO-d , TMS): 66.4 (5”CH), 110.2 (C-7),

2

6

10.5 (C-3), 111.4 (C-4), 116.9 (C-6), 124.5 (2’,6’CH), 127.3 (4’CH), 127.9 (CH-C (q) fused

carbon), 128.2 (3’,5’CH), 130.0 (C-2), 133.1 (1’CH), 134.5 (C-N (q) fused carbon), 160.6

6”CH), 161.2 (C-5), 172.7 (C=O), 185.2 (C=S); MS (70 ev): m/z = 383.11 (M+1); Anal. Calcd.

for C H FN OS (382.41): C, 56.53; H, 3.95; N, 21.98, Found, C, 56.60; H, 3.90; N, 21.92.

(

1

8

15

6

4

.1.9.3.

indole-3-carboxamide (9c)

Yield: 82%; m.p. 180-184 ºC; IR (KBr) υ (cm ): 3310 (NH str, br), 3094 (C-H Ar str), 1667

1-(5-amino-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)-5-nitro-2-phenyl-1H-

-1

(

C=O str), 1625 (N-C=N str), 1568 (Het. Triazine str), 1535 (C=C Ar str), 1325 (NO

2

str);

1

HNMR (300 MHz, DMSO-d ) δ: 3.98 (s, 1H, 5”CH), 4.21 (brs, 2H, 5”NH , D O exchangeable),

6

2

4

7

1

.82 (brs, 2H, CONH

.37 (m, 3H, 3’,4’,5’CH, J = 4.9 Hz, J = 8.5 Hz), 7.72 (d, 2H, 2’,6’CH, J = 7.29 Hz), 7.93 (d,

H, 7CH, J = 8.4 Hz), 8.69 (s, 1H, 4CH), 9.01 (brs, 1H, 4”NH, D O exchangeable), 12.24 (brs,

H, 2”NH, D O exchangeable); CNMR δ (ppm) (DMSO-d , TMS): 66.4 (5”CH), 108.7 (C-7),

2 2

, D O exchangeable), 7.20 (dd, 1H, 6CH, J = 2.2 Hz, J = 8.1 Hz), 7.25-

2

1

3

2

6

2

0

ACCEPTED MANUSCRIPT

1

10.6 (C-3), 116.6 (C-6), 124.2 (2’,6’CH), 127.0 (4’CH), 127.4 (C-4), 128.0 (CH-C (q) fused

carbon), 128.5 (3’,5’CH), 130.4 (C-2), 132.6 (C-5), 133.5 (1’CH), 140.2 (C-N (q) fused carbon),

61.0 (6”CH), 172.4 (C=O), 184.9 (C=S); MS (70 ev): m/z = 410.25 (M+1); Anal. Calcd. for

C H N O S (409.42): C, 52.80; H, 3.69; N, 23.95, Found, C, 52.87; H, 3.60; N, 24.01.

1

18

15

7

3

4

.1.9.4.

1-(5-amino-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)-2-phenyl-5-

trifluoromethyl)-1H-indole-3-carboxamide (9d)

(

-1

Yield: 76%; m.p. 195-199 ºC; IR (KBr) υ (cm ): 3311 (NH str, br), 3097 (C-H Ar str), 1661

(

C=O str), 1630 (N-C=N str), 1560 (Het. Triazine str), 1526 (C=C Ar str), 1174 (C-F str);

1

HNMR (300 MHz, DMSO-d ) δ: 3.95 (s, 1H, 5”CH), 4.25 (brs, 2H, 5”NH , D O exchangeable),

6

2

4

.75 (brs, 2H, CONH , D O exchangeable), 7.22-7.38 (m, 3H, 3’,4’,5’CH, J = 4.7 Hz, J = 8.4

2 2

Hz), 7.51 (dd, 1H, 6CH, J = 2.0 Hz, J = 7.9 Hz), 7.73 (d, 2H, 2’,6’CH, J = 7.24 Hz), 7.85 (d, 1H,

7

2

1

CH, J = 8.0 Hz), 8.84 (s, 1H, 4CH), 9.05 (brs, 1H, 4”NH, D O exchangeable), 12.22 (brs, 1H,

2

1

3

2 6

”NH, D O exchangeable); CNMR δ (ppm) (DMSO-d , TMS): 66.7 (5”CH), 110.7 (C-3),

11.4 (C-7), 115.0 (C-6), 117.4 (C-4), 124.0 (2’,6’CH), 124.6 (CF ), 125.3 (C-5), 127.5 (4’CH),

3

28.4 (CH-C (q) fused carbon), 128.7 (3’,5’CH), 130.5 (C-2), 133.0 (1’CH), 134.7 (C-N (q)

fused carbon), 161.3 (6”CH), 172.3 (C=O), 185.2 (C=S); MS (70 ev): m/z = 433.03 (M+1);

Anal. Calcd. for C19

N, 19.39.

15 3 6

H F N OS (432.42): C, 52.77; H, 3.50; N, 19.43, Found, C, 52.83; H, 3.42;

4

.1.9.5.

1-(5-amino-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)-5-methoxy-2-phenyl-

H-indole-3-carboxamide (9e)

1

-1

Yield: 80%; m.p. 177-179 ºC; IR (KBr) υ (cm ): 3309 (NH str, br), 3090 (C-H Ar str), 2971 (C-

H str), 1668 (C=O str), 1623 (N-C=N str), 1569 (Het. Triazine str), 1533 (C=C Ar str), 1255 (C-

1

O-C str); HNMR (300 MHz, DMSO-d ) δ: 3.70 (s, 3H, OCH ), 4.00 (s, 1H, 5”CH), 4.20 (brs,

6

3

2

H, 5”NH , D O exchangeable), 4.97 (brs, 2H, CONH , D O exchangeable), 7.08 (dd, 1H, 6CH,

2 2 2 2

J = 2.0 Hz, J = 8.4 Hz), 7.24-7.38 (m, 3H, 3’,4’,5’CH, J = 4.6 Hz, J = 8.5 Hz), 7.64 (s, 1H,

CH), 7.68 (d, 2H, 2’,6’CH, J = 7.25 Hz), 7.82 (d, 1H, 7CH, J = 8.5 Hz), 9.03 (brs, 1H, 4”NH,

4

1

3

D O exchangeable), 12.23 (brs, 1H, 2”NH, D O exchangeable); CNMR δ (ppm) (DMSO-d ,

2

6

TMS): 55.4 (OCH ), 66.3 (5”CH), 101.8 (C-4), 110.3 (C-7), 110.7 (C-3), 112.3 (C-6), 124.1

3

(

2’,6’CH), 127.2 (4’CH), 127.6 (CH-C (q) fused carbon), 128.2 (3’,5’CH), 130.5 (C-2), 133.5

1’CH), 134.3 (C-N (q) fused carbon), 149.3 (C-5), 161.0 (6”CH), 172.9 (C=O), 185.3 (C=S);

18 6 2

MS (70 ev): m/z = 395.32 (M+1); Anal. Calcd. for C19H N O S (394.45): C, 57.85; H, 4.60; N,

2

1.31, Found, C, 57.90; H, 4.66; N, 21.37.

4

.1.9.6.

1-(5-amino-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)-5-(methylthio)-2-

phenyl-1H-indole-3-carboxamide (9f)

Yield: 84%; m.p. 194-198 ºC; IR (KBr) υ (cm ): 3315 (NH str, br), 3096 (C-H Ar str), 2966 (C-

H str), 1665 (C=O str), 1628 (N-C=N str), 1572 (Het. Triazine str), 1528 (C=C Ar str); HNMR

-1

1

(

300 MHz, DMSO-d ) δ: 2.42 (s, 3H, SCH ), 3.97 (s, 1H, 5”CH), 4.22 (brs, 2H, 5”NH , D O

6 3 2 2

2

1

ACCEPTED MANUSCRIPT

exchangeable), 5.00 (brs, 2H, CONH

.5 Hz, J = 8.3 Hz), 7.48 (dd, 1H, 6CH, J = 1.9 Hz, J = 7.9 Hz), 7.73 (d, 2H, 2’,6’CH, J = 7.30

Hz), 7.82 (d, 1H, 7CH, J = 8.3 Hz), 7.91 (s, 1H, 4CH), 9.03 (brs, 1H, 4”NH, D O exchangeable),

2.24 (brs, 1H, 2”NH, D O exchangeable); CNMR δ (ppm) (DMSO-d , TMS): 15.8 (SCH ),

2 2

, D O exchangeable), 7.27-7.40 (m, 3H, 3’,4’,5’CH, J =

4

2

1

3

1

6

1

2

6

3

6.8 (5”CH), 110.4 (C-3), 111.6 (C-7), 112.3 (C-6), 119.6 (C-4), 124.7 (2’,6’CH), 127.1 (4’CH),

27.8 (CH-C (q) fused carbon), 128.3 (3’,5’CH), 130.6 (C-2), 132.4 (C-5), 133.6 (1’CH), 134.3

(

C-N (q) fused carbon), 161.1 (6”CH), 172.6 (C=O), 185.5 (C=S); MS (70 ev): m/z = 411.08

M+1); Anal. Calcd. for C H N OS (410.52): C, 55.59; H, 4.42; N, 20.47, Found, C, 55.66; H,

19

18

6

2

4

.37; N, 20.40.

4

.2. Pharmacology

.2.1. Anticonvulsant screening

In these studies adult male albino mice weighing (20 ± 2 g) and adult wistar rats (180 ± 10 g)

obtained from the Central Animal House Facility, (173/CPCSEA, 28 Jan., 2000) Hamdard

(

University, New Delhi-110062) were used throughout. Animals were housed in groups of 4-5

and were allowed free access to food pellets and water except for the short time that animals

were removed from their cages for testing. They were allowed to acclimatize with for a 24 h

period before testing. All behavioural experiments were conducted during the period between

1

0:00 and 13:00 with normal room light (12 h regular light/dark cycle) and temperature (26 ± 2

º

C). Animals housing and rearing had been followed optimum standard rules. Unnecessary

disturbance of animals was avoided. Animals were treated gently; squeezing, pressure and tough

manoeuvre is avoided. Instruments used for drug preparation and animal injection were

previously sterilized. All the experimental protocols were carried out with the permission from

Institutional Animal Ethics committee (IAEC) for animal care and use as follows:

4

.2.1.1. Preliminary anticonvulsant screening

All the synthesized compounds were screened for their potential in vivo anticonvulsant activity.

The preliminary pharmacological screening involves MES and scPTZ assays. The Electro-

convulsometer with ear clip electrode was used for the assessment of anticonvulsant potential

against electroshock induced seizures. Neurotoxicity was ascertained by Minimal motor

impairment test using rotarod. Four animals (swiss albino mice) were selected for each treatment

and standard group. Phenytoin and ethosuximide were used as standards during the

intraperitoneal administration of the test compounds [44]. The active compounds used for oral

investigation (100 mg/kg) and the reference drug phenytoin (30 mg/kg, MES test) and sodium

valproate (200 mg/kg, scPTZ test) [45,47] suspended in a vehicle (0.5% sodium

carboxymethylcellulose) were administered to a group of six mice each 60 min before the

injection of PTZ and MES test. The control animals were administered the vehicle. The

2

ACCEPTED MANUSCRIPT

procedures were carried out as described. One way analysis of variance (ANOVA) followed by

Dunnett’s method with the aid of Graph pad Prism software, version 5 (inc., San Diego, USA)

was used for statistical analysis.

4

.2.1.1.1. Maximal Electroshock test (MES test)

In MES test [41] mice were pre-screened 24 hour before by delivering maximal electroshock

50mA; 60Hz & 0.2s duration) by means of corneal electrodes. A drop of 0.9% Sodium chloride

(

was instilled in each eye prior to the application of electrodes in order to prevent death of the

animal. The test compounds were suspended in 0.5% sodium carboxymethylcellulose-water

mixture. The test solutions of each compound at three doses (30, 100, and 300 mg/kg body mass)

were administered intraperitoneally and the anticonvulsant activity was assessed after 0.5 h and

4

.0 h intervals respectively. Abolition of hind limb tonic extensor component of the seizure in

half or more of the animals was defined as protection. For the oral evaluation of the active

compounds maximal electric shock of 150 mA current for 0.2 s was applied through corneal

electrodes to induce convulsions and duration of hind limb tonic extension was noted as the

outcome [45] (Table 2). The abolition or reduction of the duration of hind limb tonic extension

of compounds was expressed as the mean ± S.E.M. for each group.

4

.2.1.1.2. sub-cutaneous Pentylenetetrazole test (scPTZ test)

In sc PTZ test [42] a dose of pentylenetetrazol 70 mg/Kg was utilized. This produced clonic

seizure in >95% of animals lasting for a period of atleast 5 seconds. The test compounds were

administered at the three graded doses viz. 30, 100 and 300 mg/Kg intraperitoneally. At the

anticipated time the convulsant was administered as 0.5% solution subcutaneously in the

posterior midline. Animals were observed over a 30 min period. Absence of clonic spasm in half

or more of the animals in the observed time period indicates a compounds ability to abolish the

effect of pentylenetetrazol on seizure threshold. During the oral evaluation of the active

compounds against PTZ (85 mg/Kg, sc) induced seizures, each animal was placed into an

individual plastic cage for observation over a period of 20 min. Seizure latency was defined as

the time elapsed from the injection of pentylenetetrazole to the first two myoclonic jerks of the

forelimbs. Animals devoid of generalized convulsions were considered to be protected and the

results were represented as percentage protection [46] (Table 2).

4

.2.1.1.3. Minimal motor impairment test (Rotarod test)

The minimal motor impairment is measured in mice by rotarod test [43]. The mice were trained

to stay on an accelerating rotarod of diameter 3.2 cm that rotates at 10 rpm. The animals were

injected intraperitoneally with the test compounds at a dose of 30, 100 and 300 mg/kg in a group

of four mice each. Neurotoxicity was indicated by the inability of the animal to maintain

equilibration on the rod for atleast one minute in each of the three trials. The dose at which 50%

of the animals enabled to balance themselves and fall off the rotating rod was determined. The

2

3

ACCEPTED MANUSCRIPT

active candidates were given orally (100 mg/kg) in the mice for neurotoxicity investigation

following the similar evaluation procedure as mentioned above.

4

.2.1.2. Quantitative anticonvulsant study

The quantification study of the selected compounds for the determination of median effective

dose (ED ) and median neurotoxic dose (TD ) was done using the literary procedure. For the

5

0

50

determination of ED and TD values, groups of six mice were given a range of intraperitoneal

5

0

50

doses of the test drug until at least three points were established in the range of 10–90% seizure

protection or minimal observed neurotoxicity. From the plot of these data, the respective ED₅₀

and TD values, 95% confidence intervals, slope of the regression line, and the standard error of

5

0

the slope were calculated by means of the computer program [48].

4

.2.2. Isolation of Rat Brain Regions and GABA Assay

The GABA assay was performed in brain tissue extracts enzymatically. Adult Wistar rats were

divided into three groups of six animals each. After 2 h of drug administration (100 mg/kg, i.p),

the animals were decapitated and the brains were dropped into separate vials containing 4-6 mL

of ice-cold 80% ethanol and processed further as described in reference [50]. A chronic study

was also carried out after p.o administration of the test compounds (30 mg/kg) for 7 days.

4

.2.3. Sodium Channel Binding Assay

The procedure was similar to reported methods [51]. Synaptoneurosomes were prepared from rat

cerebral cortex as follows: cerebral cortex (approximately 1-g weight) was homogenized in 2 mL

of buffer containing 130 mM choline chloride, 50 mM HEPES [adjusted to pH 4 with tris-

(hydroxymethyl)aminomethane, approximately 23 mM Tris base], 5.5 mM glucose, 0.8 mM

MgSO and 5.4 mM KCl. The tissue was homogenized with 10-12 strokes of a glass-glass

4

homogenizer. The final volume was adjusted to 6 mL and the preparation centrifuged at 1000 g

for 15 min at 4 °C. The pellet was resuspended in a total volume of 20 mL of HEPES buffer for

binding studies. Incubations were carried out for 40 min at 25 °C in a total volume of 320 ꢀL

3

containing 10 nM [ H]BTX-B, 50 ꢀg/mL of scorpion venom, approximately 980 ꢀg of the

particulate vesicular protein, and varying concentrations of added test compound (from 50 mM

stock solutions in 50% MeOH/H,O). The MeOH concentration was in all cases less than 1 %.

Incubations were terminated by dilution of the reaction mixture with 3 mL of ice-cold wash

buffer and filtration through a Whatman GF/C filter paper. Filters were washed with wash buffer

(

3 x 3 mL). The wash buffer contained the following: 163 mM choline chloride, 5 mM HEPES

adjusted to pH 7.4 with Tris base), 1.8 mM CaCl , and 0.8 mM MgSO . Filters were counted in

2

4

a Beckman scintillation counter using 10 mL of 3270B counting cocktail. Specific binding was

determined by subtracting the nonspecific binding, measured in the presence of 250 ꢀM

3

veratridine, from the total binding of [ H]BTX-B. Specific binding was about 80% of total

binding. All experiments were performed in triplicate. IC50, values (concentration of compound

required to inhibit 50% of specific neurotoxin binding) were determined from a dose-response

2

4

ACCEPTED MANUSCRIPT

curve generated by plotting the log of anticonvulsant concentration versus percent of specifically

3

bound [ H]BTX-B.

Acknowledgement

The authors extend their gratitude Jamia Hamdard, New Delhi, India for providing research facilities, IIT, New

Delhi for spectral data and ACBR, New Delhi for in vitro sodium channel binding assay.

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Anticonvulsant evaluation of clubbed indole-1,2,4-triazine derivatives: A synthetic approach

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